Bilateral neck swelling (edema) in an elderly man

J R Soc Med. 2002 October; 95(10): 503–505.
Bilateral neck swelling in an elderly man

M K Harkness, MRCP and C K Biswas, FRCPDepartment of Medicine for the Elderly, Dewsbury & District Hospital, Dewsbury, W Yorkshire WF13 4HS, UK

Correspondence to: Dr MK Harkness E-mail: mharkness@doctors.org.uk

Swellings of the neck can present a difficult diagnostic challenge, especially when bilateral.
Case History

A domiciliary visit was requested on an 81-year-old male smoker because of weight loss, shortness of breath and general weakness. He had lately been in hospital elsewhere with pneumonia. The most striking feature on physical examination was the massive swelling on either side of his neck (Figure 1). For 25 years he had had swelling of the right cheek and for 9 years swelling of the left cheek. These caused little discomfort; both had become progressively larger over the past year. The swellings were cystic, with a little tenderness to pressure on the right. A core biopsy was attempted on both lesions, but only purulent fluid was obtained. Cultures were negative. Cytology showed no malignant cells; otherwise the samples were poorly cellular.

Ultrasound of the neck showed that the swellings were predominantly cystic, and uniformly low-level echoes suggested proteinaceous content. On a CT scan, well-defined cystic masses on each side of the neck were seen to arise from behind the angle of the mandible to the level of the hyoid (Figure 2). There was no cervical adenopathy. The neck swellings were thought to be longstanding branchial cysts. Unfortunately, radiological imaging showed metastatic disease within the liver from a primary bronchogenic carcinoma. Also, ultrasound suggested a lesion in one kidney. The patient's condition deteriorated rapidly and he died peacefully in hospital. At necropsy two primary tumours were found—a poorly differentiated non-small-cell bronchogenic carcinoma with hepatic metastases, and a small renal cell carcinoma. The neck cysts were confirmed as branchial, measuring 13×9×5 cm on the left and 17×10×8 cm on the right. On histological examination the walls of the cysts contained abundant lymphoid tissue with an attenuated lining of squamous epithelium; there were many cholesterol clefts.

Comment

There are reports of branchial cysts up to the age of 60 years1, but we have found no record of bilateral branchial cysts in older patients. These cysts emerge in the anterior triangle of the neck under the anterior border of sternocleidomastoid where the upper third meets the middle third. They present most commonly in males, usually on the left side, after the age of 10 years and peaking in the third decade of life. The cyst fluid contains cholesterol crystals.

There are several theories about the origin of branchial cysts. They may represent remnants of pharyngeal pouches or branchial clefts or they could be due to the non-disappearance of the cervical sinus (where the second branchial arch grows down over the third and fourth). Most are lined by squamous epithelium and have lymphoid tissue in the wall—hence the notion that cyst epithelium arises from lymph node squamous epithelium2.

A thorough history and examination will provide clues to the diagnosis but radiological imaging, fine-needle aspiration and core biopsy may be required3. The differential diagnosis of swelling in the neck lies between a parotid tumour, lymphadenopathy, thyroid disease, cystic hygroma, branchial cyst and a carotid body tumour. Ultrasound of a branchial cyst shows a uniformly low echogenicity4 that distinguishes these lesions from other tumours and allows prompt surgical treatment. If a parotid mass is suspected a CT scan is advisable, to define the extent of the mass and whether there is involvement of the deep lobe or the parapharyngeal space5.

MRI provides the best information because of greater tissue contrast resolution and multiplanar images1. Fine-needle aspiration and examination of the aspirate may be used to differentiate conditions that clinically mimic each other—for example, cystic nodal metastasis6. If an aspirate sample is insufficient or non-diagnostic, a core biopsy will be required. Treatment is by excision.

Acknowledgments

We acknowledge the help of Dr BA Cadman, Department of Histopathology, Dewsbury and District Hospital; the Department of Medical Illustration, Huddersfield Royal Infirmary; and Mrs L Prescot.
References

1.
McClure MJ, McKinstry CS, Stewart R, Madden M. Late presentation of branchial cyst. Ulster Med J 1998;67: 129-31 [PubMed].

2.
Chionh EH, Pham VH, Cooke RA, Gough IR. Aetiology of branchial cysts. Austr NZJ Surg 1989;59: 949-51.

3.
Smith OD, Ellis PDM, Bearcroft PWP, Berman LH, Grant JW, Jani P. Management of neck lumps—a triage model. Ann R Coll Surg Engl 2000;82: 223-6 [PubMed].

4.
Reynolds JH, Wolinski AP. Sonographic appearances of branchial cysts. Clin Radiol 1993;48: 109-10 [PubMed].

5.
Golding S. Computed tomography in the diagnosis of parotid tumours. Br J Radiol 1982;55: 182-8 [PubMed].

6.
Hardee PS, Hutchinson IL. Solitary nodal metastases presenting as branchial cysts: a diagnostic pitfall. Ann R Coll Surg Engl 1999;81: 296-8.

Journal of the Royal Scoiety of Medicine

Intraoperative Arthroscopic Cold Irrigation Solution Does Not Affect Postoperative Pain and Edema

A. Louise Fincher,* G. William Woods,† and Daniel P. O'Connor‡

*University of Texas at Arlington, Arlington, TX
†University of Texas Medical School–Houston, Texas Orthopedic Hospital, Houston, TX
‡Joe W. King Orthopedic Institute, Houston, TX

Corresponding author.

A. Louise Fincher, EdD, ATC, contributed to conception and design; acquisition and analysis and interpretation of the data; and drafting, critical revision, and final approval of the article. G. William Woods, MD, contributed to acquisition of the data, critical revision, and final approval of the article. Daniel P. O'Connor, PhD, ATC, PT, contributed to acquisition and analysis and interpretation of the data and drafting, critical revision, and final approval of the article.

Address correspondence to A. Louise Fincher, EdD, ATC, Department of Kinesiology, University of Texas at Arlington, Box 19259, Arlington, TX 76019-0259. Address e-mail to lfincher@uta.edu.

Abstract

Objective:

To determine what effect using a cold irrigating solution during arthroscopic knee surgery would have on postoperative pain intensity, pain-medicine consumption, and knee joint swelling.

Design and Setting:

We employed a randomized, controlled trial design. Subjects were randomly assigned to either the cold (4°C) irrigating saline group or room-temperature (18°C) irrigating saline (control) group. Subjects were blinded to group assignment. All surgeries were performed at the same hospital by the same surgeon.

Subjects:

The sample was 93 physically active patients (32 women, 61 men, mean age = 47.4 ± 15.1 years) who had knee injuries requiring surgery. Those with cold sensitivities or contraindications to the use of cold were excluded.

Measurements:

A 10-cm horizontal visual analog scale was used to measure postoperative pain intensity. Postoperative pain-medicine consumption was recorded using a daily log. Knee joint swelling (girth) was measured at midpatella and 2 in (5.08 cm) above midpatella. Pain and swelling measures were collected before and after surgery.

Results:

No statistical or clinical differences were found between the cold-saline and control groups for pain, pain-medicine consumption, and postoperative swelling across the first 4 postoperative days.

Conclusions:

Our results suggest that using intra-articular cold saline to irrigate the knee joint during arthroscopic surgery had no statistically or clinically significant effect on postoperative pain, medication usage, or swelling in the first 4 postoperative days.

Keywords:

cryotherapy, knee, controlled trial

Physically active persons are usually eager to return to activity after arthroscopic knee surgery. Return to activity, however, is often delayed by the pain and swelling experienced after knee surgery. Postoperative pain and swelling often inhibit quadriceps function, which may delay the initiation and progression of rehabilitation. Clinicians continually seek methods for improving the management of postoperative pain and swelling in an effort to hasten return to normal activity and improve outcomes. Cryotherapy is a very common postoperative intervention used to relieve postoperative pain and swelling; however, researchers have failed to consistently demonstrate its therapeutic effectiveness.1–6 Most of the reported inconsistencies associated with postoperative cryotherapy can be attributed to one or more of the following factors: (1) the variation in methods used to apply postoperative cold, (2) the temperatures used, (3) the length of time between surgery and initial cold application, (4) the duration and frequency of cold applications, and (5) the thickness of surgical dressings over which cold was applied. Each of these factors can significantly influence the therapeutic effectiveness of cryotherapy7 and make comparisons among studies difficult.

Dahlstedt et al8 reported that skin temperature had to be lowered to about 20°C (68°F) to produce demonstrable changes in knee intra-articular temperatures. These findings suggest that for cryotherapy to effectively reduce postoperative pain and swelling, which is believed to stem from trauma to the intra-articular tissues, skin temperatures should be reduced to at least 20°C. This magnitude of change in skin temperature is difficult to accomplish in the postoperative setting because of the barrier of protective bandaging.9,10 Of the authors who have reported skin temperatures resulting from postoperative cryotherapy, none have attained temperatures as low as 20°C.2,4

Our purpose was to examine what effect, if any, intraoperative cryotherapy during arthroscopic knee surgery would have on postoperative pain intensity, pain-medicine consumption, and knee joint swelling. Intraoperative cryotherapy enables therapeutic cold to be applied directly to the intra-articular tissues, thus regulating the temperature of the specific target tissue. Our study was based on the theoretic assumptions that postoperative pain and swelling are attributable to surgical injury of intra-articular tissues and lowering the metabolism of the intra-articular tissues during surgery would decrease those tissues' need for oxygen during and immediately after surgery.7 This would, in turn, decrease the amount of secondary hypoxic injury produced during surgery. Decreased tissue damage should lead to lower tissue oncotic and capillary filtration pressures that should, in turn, lead to less edema formation.7 Lessened tissue damage and edema should also effectively reduce the amount of postoperative pain.

Methods

Experimental

DesignIn this randomized, controlled trial, subjects were randomly assigned by coin toss to either the cold saline group (4°C) or the control group (18°C, room-temperature saline). Subjects were blinded to treatment group assignment, but the surgeon and principal investigator were not. The dependent variables were postoperative pain intensity, pain-medicine consumption, and knee joint swelling (girth). Approval for the use of human subjects was obtained from the facility's institutional review board.
We conducted an a priori power analysis to estimate the sample size required for detecting a large effect (≥0.10).11 With alpha set at 0.05 and power equal to 0.80, a sample size of 17 patients per group was calculated. Because there were 3 dependent variables, the calculated sample size was tripled to ensure adequate power. An additional 10% was added to the 51 subjects per group calculation to counter potential subject attrition, for a final minimum group size of 56 subjects.

Subjects

We recruited 122 consecutive patients who had a knee injury requiring arthroscopic surgery from the orthopaedic practice of a study coinvestigator. Patients requiring an arthroscopically assisted anterior cruciate ligament reconstruction or a lateral retinacular release were excluded. A Cold Sensitivity Questionnaire was created for this study and used to screen all potential subjects for sensitivities or conditions that might contraindicate the use of cold.1,12–15 Patients without known cold sensitivities or contraindicating conditions consented to participate in this study by signing the informed consent form. Informed consent was provided by the parents of subjects who were minors (under age 18 years). All subjects were given an explanation of the study procedures and the potential risks to participants.

Instrumentation and Procedures

The principal investigator met with patients on the morning of their surgery to collect baseline pain and swelling measures. A 10-cm horizontal visual analog scale (VAS) with verbal anchors of “no pain” and “worst pain possible” was used to measure postoperative pain intensity.16

Subjects were instructed to make a single vertical mark that crossed the VAS at the point between the 2 extremes that represented their peak pain over the previous 24 hours. The VAS has been widely reported to be reliable and valid for measuring pain intensity17,18 and has been specifically recommended for measuring pain during recovery from knee surgery.17,19 Before surgery, subjects completed their baseline VAS rating of knee pain intensity. Subjects were also given a packet containing 4 additional VAS instruments and were instructed to complete them once per day at 6 hours (day 1), 24 hours (day 2), 48 hours (day 3), and 72 hours (day 4) after surgery. This measurement schedule for the VAS followed those previously described in the literature for patients with postoperative knee pain.17,19 Subjects were instructed how to record their prescription pain medication consumption (either hydrocodone bitartrate 5 mg and acetaminophen 500 mg or codeine phosphate 30 mg and acetaminophen 500 mg; both are schedule III narcotics) using a preprinted pain-medicine log for postoperative days 1 through 4. Quantifying postoperative pain-medicine consumption provides a reliable, simple, objective, and reproducible indication of pain.20

A Lufkin tape measure with a Gulick spring-loaded attachment (Cooper Industries, Inc, Houston, TX) was used to assess knee joint girth. Girth measures were collected for both knees at midpatella and 2 in (5.8 cm) above midpatella. Side-to-side comparative girth measures were used as indicators of knee joint swelling. A swelling score was calculated for each measurement site (surgical knee [cm]–nonsurgical knee [cm]), and the swelling scores for each site were summed for an overall rating of swelling.

After instructions and baseline measures, the subjects were randomly assigned by coin toss to either the cold-saline group or the control group. Making the random assignment after collecting the baseline measures ensured that group assignment did not bias the delivery of instructions to subjects. The subject's group assignment was documented on the surgical schedule to enable the circulating nurses to prepare and hang the appropriate bags of irrigating saline.

All surgeries were performed at the same hospital by the same surgeon (a study coinvestigator). All arthroscopies were done on an outpatient basis using general anesthesia. The gravity method was used to administer a continuous flow of the saline irrigating solution during all the arthroscopic procedures. The circulating nurse monitored the level of saline throughout the case and hung additional bags of the appropriate solution as needed.

Bags of normal saline were cooled to 4°C (39.2°F) in 2 commercial-grade, hazardous-location refrigerators (Grainger, Houston, TX) that were located in the substerile storage room just outside the operating room suite. The temperatures of the refrigerators and the saline bag supply were checked daily. Two neoprene sleeves were made to insulate the bags of saline during arthroscopic surgery. The sleeves were constructed such that they could easily be slipped onto the bags of saline before hanging, maintaining the temperature of the cold saline during the surgical procedure. The bags of room-temperature saline (18°C [64.4°F]) were stored on a shelf in the same substerile storage room.

Knee intra-articular temperature was recorded at the beginning and end of every arthroscopic procedure using a sterile temperature probe and digital temperature monitor (Mallinckrodt, Inc, Irving, TX). To standardize the procedure, the temperature probe was inserted through the anterior portal and directed such that the tip rested in the anterior intercondylar notch.21 The probe was held in this position for 1 minute, and then the temperature reading was recorded. The first measurement was taken as soon as the portal incisions were made and the arthroscopic instruments were inserted, providing a clear view of the intercondylar notch. The second and final measurement was taken at the conclusion of the surgical procedure, immediately preceding removal of the arthroscopic instruments from the knee.

At the conclusion of all arthroscopic procedures, subjects received an intra-articular injection of bupivacaine (20 mL, 0.5% with epinephrine, 1:200000) and morphine (1 mL, 10 mg). This routine was standard practice for all arthroscopic procedures performed by the coinvestigator and was therefore not changed for this study. All patients were instructed to bear weight as tolerated, to use crutches as necessary for balance, and to maintain a normal gait pattern (ie, without a quadriceps- avoidance gait22). Patients were instructed in quadriceps-setting exercises, straight-leg raises, and active knee flexion (sitting with the foot of the surgical leg sliding under the chair). Most patients attended at least 2 to 3 rehabilitation sessions on consecutive postoperative days in an outpatient clinic or athletic training room. Patients were informed that they could use ice packs for 15 minutes several times a day, but that this was not required. Neither the surgeon nor the principal investigator had any contact with the subjects between their surgery and first postoperative office visit (usually day 5).

The VAS instruments and pain-medicine logs were collected from the subjects at their first follow-up appointment with the surgeon. Patients who forgot to bring their forms were given a self-addressed, stamped envelope to return their forms. Of the 122 subjects recruited, 93 (32 females, 61 males) completed the study. The remaining 29 subjects returned incomplete data forms. During the first follow-up visit, the principal investigator also reassessed swelling by repeating the girth measurements (centimeters) with the same tape measure that was used for the preoperative measurements.

Statistical Analyses

We computed descriptive statistics for all variables, using means, SDs, and ranges for continuous variables and frequencies for categoric variables. Independent-samples Student t tests and χ2 tests were used as appropriate to compare the independent variables between the treatment groups. Pearson product moment correlation coefficients were calculated for all pairwise relations among VAS ratings, pain-medicine consumption, swelling, and articular temperatures. To address the primary research questions, orthogonal contrasts in a multivariate general linear-model analysis were used to test for group differences in pain (VAS ratings across 4 days), pain- medicine consumption, and swelling. Our 3 hypotheses were tested independently of one another and therefore did not require alpha value corrections, as would be necessary with multiple (dependent) comparisons or tests.

Results

No significant differences were seen in pain (P = .385), pain-medicine consumption (P = .989), or postoperative swelling (P = .966) between the treatment groups. There was no evidence that use of the cold saline irrigating solution during arthroscopy of the knee had any effect on postoperative pain, medication use, or swelling.

The treatment groups did not differ by age (P = .652) or sex distribution (P = .392, Table 1). Surgical procedures by treatment group are presented in Table 2. Intra-articular temperature between treatment groups was significantly different (P < .0005), indicating the cold saline had cooled the knee as intended. The mean intra-articular knee temperature for the cold saline group (7.7°C) was 12.1°C colder than the control group (19.8°C).

The mean pain rating across the 4 postoperative days was significantly correlated with pain-medicine consumption across (r = 0.29, P = .005) and within groups (rCold = 0.28, rControl = 0.30; Table 3). Pain-medicine consumption increased slightly as pain ratings increased, and medication use decreased slightly as pain ratings decreased. Postoperative swelling (increase in girth relative to baseline measure) was not significantly correlated with pain ratings (r = 0.11, P = .280). Intra-articular knee temperature readings (beginning and end) were also uncorrelated with the pain ratings.

Discussion

Our results suggest that intra-articular cold saline irrigation during arthroscopic knee surgery had no statistically or clinically significant effect on postoperative pain, medication intake, or swelling. Several potential explanations for the lack of effect exist. First, intra-articular tissue insult may not have been the primary cause of postoperative pain associated with these arthroscopic knee surgeries. The extra-articular tissue insult caused by insertion of the arthroscopic instruments, however, may have created an inflammatory response. The intra- articular cold saline irrigation probably did not alter the temperature of the superficial knee tissues (skin, superficial muscle, etc) and thus would not have affected the signs and symptoms associated with injury of those tissues. Many of the intra-articular procedures did not involve the synovium and therefore may have produced little to no intra-articular inflammation.

Alternately, the cold saline may have been withdrawn from the intra-articular tissues before any inflammatory response had begun. Most of the arthroscopic procedures were less than 30 minutes in duration. The first portion of each procedure consisted of diagnostic arthroscopy of the knee joint. The surgical tissue insults occurred in the latter part of each procedure, just before closing. In many cases, the cold saline irrigation continued for less than 10 minutes after conclusion of the surgical tissue insult (eg, partial meniscectomy). If the knees had continued to be irrigated with cold saline for 10 or 15 minutes after conclusion of the surgical procedure, an effect on one or more of the postoperative measures might have been seen. Unfortunately, the material and financial costs associated with extending operating room time are likely to outweigh any potential benefit that may result from continuing the cold saline irrigation after the conclusion of the procedure.
It is also possible that the treatment provided in the outpatient clinics and athletic training rooms varied somewhat among patients and may have contributed to the lack of an effect. For example, patients with more swelling and pain may have received more intensive treatment to address these problems. The duration, intensity, and specific activities of postoperative rehabilitation were not controlled or measured in this study because there was no reliable way of observing them. We suspect that, as a whole, neither group received more rehabilitative treatment because pain and swelling did not differ between groups. The prospective, randomized, controlled-trial study design we used is the most powerful research design possible for control of factors that are beyond the immediate control of the investigators.

Despite the lack of any detectable treatment effect, our results did demonstrate a few trends. Subjects within both treatment groups demonstrated a consistent pain pattern (Table 3, Figure) with peak pain intensity occurring on day 2. This pattern is similar to that reported by Brown et al23 and may be common to most patients with arthroscopic knee surgery. The intra-articular injection of bupivacaine and morphine that all subjects received at the conclusion of their surgery may have produced lower VAS scores on day 1 compared with day 2.
During their follow-up examinations, several subjects provided unsolicited, anecdotal comments regarding their postoperative pain. These comments ranged from “I know I must have received the cold treatment because I really didn't have much pain” to “I'm pretty sure I didn't get the ‘cold stuff’ since my knee really hurt pretty bad.” After recording each set of anecdotal comments, the principal investigator compared the subject's pain ratings with his or her comments. In no case did the subject's comments and the qualitative interpretation of the pain ratings match. For example, subjects who commented that they had experienced very little pain often produced VAS scores that would be interpreted as moderate to severe (VAS rating = >31 mm24,25).

This empirical inconsistency suggests that the single VAS rating per day may not have been representative of the subjects' pain. Jensen and McFarland26 stated, “A single rating of pain intensity is not adequately reliable or valid as a measure of average pain” for persons with chronic pain and recommended that VAS ratings should occur every 2 hours or at least 6 times per day. They suggested that an average of these measures would provide a more reliable and accurate measure of pain. It is not known whether this measurement schedule would be appropriate for persons with acute postsurgical pain. We employed a once-a-day measurement schedule based on currently accepted methods for this population.17,19

The use of cold saline produced a very small effect among our subjects. The a priori power analysis to determine sample size was estimated using an effect size of at least 0.10 (ie, cold saline would explain at least 10% of the differences between treatment groups). The largest observed effect size in our analyses was 0.008, corresponding to a mean group difference of only 0.34 cm on the 10-cm VAS scale. The very small effect sizes in the current study were much less than what we had designated as being clinically relevant.

Chilled saline has been shown to be successful in reducing pain in neurosurgical procedures.27,28 For this reason, the therapeutic effects of intraoperative cold applications should not be discounted for orthopaedic procedures. Future researchers in this area may consider avoiding the use of intra-articular medication immediately after the procedure and collecting more than one VAS reading each day to increase reliability and responsiveness of pain measurement. In addition, prolonged irrigation with cold saline after completion of the procedure may coincide with onset of inflammation, thereby potentially producing a larger effect. Also, probes could be inserted directly into several tissues surrounding the knee to evaluate the degree to which cold saline irrigation lowers their temperature. Finally, we recommend that the intra-articular cold applications be tested with surgical knee procedures that involve greater intra-articular tissue insult, such as anterior cruciate ligament reconstruction.

Acknowledgments

This study was funded by a research grant (#398B004) from the National Athletic Trainers' Association Research & Education Foundation.

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Weresh MJ, Bennett GL, Njus G. Analysis of cryotherapy penetration: a comparison of the plaster cast, synthetic cast, Ace wrap dressing, and Robert-Jones dressing. Foot Ankle Int. 1996;17:37–40. [PubMed]

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Culp RW, Taras JS. The effect of ice application versus controlled cold therapy on skin temperature when used with postoperative bulky hand and wrist dressings: a preliminary study. J Hand Ther. 1995;8:249–251. [PubMed]

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Cohen, J. Statistical Power Analysis for the Behavioral Sciences. Hillsdale, NJ: Lawrence Erlbaum Assoc; 1988.

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Escher SA, Tucker AM. Preventing, diagnosing, and treating cold urticaria. Physician Sportsmed. 1992;20(12):73–84.

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Sriwatanakul K, Kelvie W, Lasagna L, Calimlim JF, Weis OF, Mehta G. Studies with different types of visual analog scales for measurement of pain. Clin Pharmacol Ther. 1983;34:234–239. [PubMed]

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Jensen MP, Chen C, Brugger AM. Postsurgical pain outcome assessment. Pain. 2002;99:101–109. [PubMed] [Full Text]

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Breivik EK, Bjornsson GA, Skovlund E. A comparison of pain rating scales by sampling from clinical trial data. Clin J Pain. 2000;16:22–28. [PubMed] [Full Text]

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Flandry F, Hunt JP, Terry GC, Hughston JC. Analysis of subjective knee complaints using visual analog scales. Am J Sports Med. 1991;19:112–118. [PubMed]

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Jaureguito JW, Wilcox JF, Cohn SJ, Thisted RA, Reider B. A comparison of intraarticular morphine and bupivacaine for pain control after outpatient knee arthroscopy: a prospective, randomized, double-blinded study. Am J Sports Med. 1995;23:350–353. [PubMed]

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Journal of Athletic Training

Edema and the tropics

Abstract

Holzer BR.

Facharzt fur Allgemeine Medizin FMH und Tropen- und Reisemedizin FMH, Mittlere Strasse 3, CH-3600 Thun.

benedikt.holzer@bluewin.ch

People visiting or living in tropical or subtropical regions are exposed to various factors, which can lead to edema. Tourists staying for only a short time in the tropics are exposed to different risks, with other disease patterns, than people living in the tropics or immigrants from tropical regions. The differential diagnosis of edema and swelling is extensive and it can sometimes be difficult to distinguish classical edema with fluid retention in the extravascular interstitial space, from lymphedema or swelling due to other aetiologies. The patients often connect the edema to their stay in the tropics although it may have been pre-existing with no obvious relation to their travels.

Already the long trip in the plane can lead to an "economy class syndrome" due to deep venous thrombosis. Contacts with animal or plant toxins, parasites or parasitic larvae can produce peripheral edema. The diagnosis can often only be made by taking a meticulous history, checking for eosinophilia and with the help of serological investigations. Chronic lymphedema or elephantiasis of the limbs is often due to blocked lymph vessels by filarial worms. It has to be distinguished from other forms as e.g. podoconiosis due to blockage by mineral particles in barefoot walking people.

The trend to book adventure and trekking holidays at high altitude leads to high altitude peripheral edema or non-freezing cold injuries such as frostbites and trench foot. Edema can be an unwanted side effect of a range of drugs e.g. nifedipine, which is used to prevent and treat high altitude pulmonary edema. Protein malnutrition, (Kwashiorkor), and vitamin B6 deficiency, (Beri-Beri) are very rarely observed in immigrants and almost never in tourists. A very painful swelling of fingers and hands in children and young adults of African origin can be observed during a sickle cell crisis.

Many protein loosing nephropathies connected with plant and animal toxins but also bacterial, viral or parasitic agents, can lead to edema. But very often edema in tourists or immigrants from the tropics is not related to their stay abroad. To take an accurate history of the itinerary, eating habits and exposure to water etc. is very important. Knowledge of the precise epidemiology and geographic distribution of diseases are essential.

Publication Types:

Case Reports

Review

PMID: 15605460 [PubMed - indexed for MEDLINE]

Anthrax Edema Toxin

Anthrax Edema Toxin Requires Influx of Calcium for Inducing Cyclic AMP Toxicity in Target Cells

Praveen Kumar, Nidhi Ahuja, and Rakesh Bhatnagar*

Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

*Corresponding author. Mailing address: Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. Phone: (91) 11-6179751. Fax: (91) 11-6198234/6865886.

E-mail: rakbhat01@yahoo.com.

Received April 9, 2002; Revised May 9, 2002; Accepted May 27, 2002.

Abstract

The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.

Anthrax, which is primarily a zoonotic disease, is transmissible from animals to humans. The causative agent of anthrax, Bacillus anthracis, is a gram-positive, spore-forming bacterium that produces a three-component exotoxin called the anthrax toxin complex (22). The three components of this toxin complex are protective antigen (PA [83 kDa]), edema factor (EF [89 kDa]), and lethal factor (LF [90 kDa]). Individually, all of the three proteins are nontoxic. However, the combination of PA and LF, called the lethal toxin, causes death in experimental animals (30), whereas the combination of PA and EF, known as the edema toxin, induces an increase in the intracellular cyclic AMP (cAMP) levels in the susceptible cells (20) and elicits skin edema upon subcutaneous injection (31). By increasing the intracellular cAMP concentrations in neutrophils, anthrax edema toxin inhibits phagocytosis and blocks both particulate as well as phorbol myristate acetate-induced chemiluminescence (25). Anthrax edema toxin can differentially regulate lipopolysaccharide-induced production of tumor necrosis factor alpha and interleukin-6 by increasing the intracellular cAMP levels in monocytes (16).

Both LF and EF require PA for their entry into target cells. During intoxication of the target cells, PA binds to the receptors on the cells surface (6). It gets cleaved by cell surface proteases, such as furin (18), to release an N-terminal 20-kDa fragment, PA20, from the cell surface, thereby exposing a high-affinity site on the 63-kDa fragment, PA63, still bound to the receptor. PA63 then binds to the catalytic components, EF or LF. The entire complex undergoes receptor-mediated endocytosis. The acidification of the endosome (13) results in the insertion of PA63 into the endosomal membrane (24) and the translocation of EF and LF into the cytosol of target cells (14), where they exert their toxic effects.

EF is an adenylate cyclase. After gaining access to the cell cytoplasm, it gets activated by calmodulin to catalyze the synthesis of cAMP in the host cells (20). Studies on the enzymatic activity of EF demonstrated that EF has a high catalytic activity with a Vmax of 1.2 mmol cAMP/min/mg of protein (21). The adenylate cyclase activity of EF is very sensitive to concentrations of calcium, showing optimal activity at 0.2 mM and inhibition at higher concentrations of calcium. Activation of EF by calmodulin is calcium dependent. In the absence or presence of 50 μM calcium, the concentrations of calmodulin giving half-maximal activity are 5 μM and 2 nM, respectively (21).

The free-calcium concentration in nonexcitable cells is kept very low (usually 10 to 100 nM). The concentration of calcium outside the cell is approximately 2.0 mM (12). Is the basal concentration of free calcium in cell cytosol enough for calmodulin-dependent activation of EF? Or does EF, like many other calcium- and calmodulin-dependent adenylate cyclases (15, 33), requires the influx of calcium for optimal activation? To address these questions in the present study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced accumulation of cAMP in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, cultured macrophages RAW264.7, and human immune effector cells, namely, neutrophils and lymphocytes, that may be physiological targets of anthrax edema toxin. Although the response generated by edema toxin in these cells varied in intensity and in the time of initiation, the dependence on extracellular calcium was a common feature. The data presented here demonstrate that, after the translocation of edema factor into the cell cytosol, increased influx of calcium is triggered in the host cell which plays a critical role in determining the ensuing cAMP response. The absence of calcium or the presence of calcium channel antagonists in the extracellular medium prevented the cAMP accumulation by edema toxin in the host cells. To the best of our knowledge, this is the first report demonstrating edema toxin-induced accumulation of cAMP in lymphocytes. By accumulating cAMP, edema toxin may be disrupting bactericidal functions of immune effector cells and disabling the host defense mechanism.

Material and Methods

Purification of edema factor. The procedures followed for the expression and purification of EF have been described in detail in a previous publication (19). Briefly, the construct, pPN-EF (containing full-length structural gene of edema factor under the control of T5 promoter), was transformed into Escherichia coli SG13009 cells. The cells were grown to an optical density at 600 nm of 0.8, induced, and later harvested. The cells were lysed and EF was purified to homogeneity using a two-step procedure involving Ni-nitrilotriacetic acid (NTA) metal-chelate affinity chromatography and SP-Sepharose cation-exchange chromatography.
Purification of protective antigen. PA was expressed and purified according to the procedures described previously (2). Briefly, E. coli BL21(DE3) cells harboring the plasmid pMS1 were induced with IPTG (isopropyl-β-d-thiogalactopyranoside) and later harvested. The periplasmic fraction was isolated, and the protein was purified to homogeneity by ResourceQ ion-exchange and phenyl-Sepharose hydrophobic interaction chromatography.

Cell culture. Macrophage-like cell line RAW264.7 was maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). The Chinese hamster ovary (CHO) cell line was maintained in Eagle minimal essential medium (EMEM) supplemented with nonessential amino acids, 25 mM HEPES (pH 7.4), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% heat-inactivated fetal calf serum. Calcium-free EMEM (Gibco) or calcium-free Hanks balanced salt solution (HBSS; Gibco) was used for cell culture assays that were performed in calcium-free medium.

Isolation of lymphocytes and neutrophils from human blood. Human lymphocytes were obtained by sedimentation of heparinized whole blood on Ficoll-Hypaque density gradient, as detailed previously (10). The procedure for isolation of neutrophils from heparanized blood involves sequential sedimentation in dextran, density centrifugation in Ficoll-Hypaque, and lysis of contaminating red blood cells by osmotic shock, as described previously (10).

Adenylate cyclase activity of edema toxin in target cells. To determine the cAMP response generated by edema toxin in cells, the cells were treated with various concentrations of edema toxin in EMEM medium (with or without calcium) at 37°C. After incubation of the cells with the toxin for the indicated period of time, the media was removed, and the cells were lysed with lysis buffer (supplied with Biotrak cAMP EIA kit) at 37°C for 10 min. The cell debris was removed by centrifugation. The supernatant was collected and was processed for the enzyme-linked immunosorbent assay-based determination of intracellular cAMP concentrations by using the nonradioactive Biotrak cAMP EIA Kit (Amersham Pharmacia).

Radioiodination of PA and EF. A total of 100 μg of the protein was allowed to react with 1 mCi of Na125I (specific activity, 17.4 Ci/mg) and 1 μg of Chloramine T in 40 μl of 0.1 M phosphate buffer (pH 7.0). After 1 min, the reaction was stopped by the addition of 5 μg of sodium metabisulphite. The labeled protein was separated from free radioactivity on a Sephadex G-25 column that was presaturated with 0.1 M phosphate buffer (pH 7.0) containing 1% bovine serum albumin (BSA). The specific activity was 1.2 × 107 cpm/μg for EF and 1.9 × 107 cpm/μg for PA.

Calcium uptake. To study the uptake of calcium (3), CHO cells were plated in 24-well plates and treated with edema toxin (12 nM PA, along with 1.1 nM EF) in EMEM containing 1 mM nonradioactive calcium (with or without the calcium channel antagonists). 45CaCl2 (5 μCi/ml) was supplemented to successive wells after every 10 min. Incubation of the cells with 45Ca2+ was allowed for 10 min in each well. The cells were then washed four times with HBSS and dissolved in 0.1 N NaOH. Radioactive counts associated with the cells were measured by scintillation counting to determine the uptake of 45Ca2+ by the cells within 10 min of incubation with 45CaCl2. The data has been expressed as percentage 45Ca2+ uptake over control.
Binding of PA to the cell surface receptors. CHO cells were plated in 24-well plates and were grown to confluence. Before the start of the experiment, the cells were washed with cold calcium-free HBSS.

Radioiodinated PA (12 nM) was then added to these cells in the presence or absence of calcium. After 30-min of incubation at 4°C, the cells were washed again to remove unbound protein. The cells were then solubilized with 0.1 N NaOH. Radioactive counts associated with the cells were measured to determine the amount of PA that bound to the cells in the presence or absence of calcium. To calculate the specific binding of PA, the difference between the mean binding of triplicate samples in the absence or the presence of a 100-fold molar excess of nonradioactive PA was taken.

To determine the effect of calcium channel antagonists on the binding of PA to the cell surface receptors, the cells were incubated with radioiodinated PA in cold medium containing calcium and 100 μM antagonist. The cells were later washed and solubilized, and the radioactivity associated with the cells was measured as detailed above.

Proteolytic activation of PA. CHO cells were allowed to grow to confluence in 24-well plates. The cells were washed, and radioiodinated PA (12 nM) was allowed to bind to the cells in the presence of calcium at 4°C. After 30 min, the cells were washed three times with cold calcium-free medium to remove unbound protein. The cells were then incubated, at 4°C, in the presence of calcium (with or without calcium channel antagonists) or in the absence of calcium to allow for the proteolytic cleavage of PA. After 90 min of incubation, the cells were lysed with sodium dodecyl sulfate (SDS) lysis buffer. The cell lysate was resolved on a SDS-12% polyacrylamide gel electrophoresis (PAGE). The gel was later dried and exposed to X-ray film. The band (corresponding to the 63-kDa fragment of PA) was cut from the gel, and the associated radioactivity was measured to determine the amount of PA that converts to the 63-kDa fragment in the presence or absence of calcium.

Oligomerization of PA and its insertion. CHO cells plated in 24-well plates were grown to confluence. The cells were washed with cold medium and radioiodinated PA (12 nM) was added to these cells in EMEM containing calcium. After incubation of the cells for an hour at 4°C, the cells were washed with cold calcium-free medium to remove unbound protein. The cells were then treated with fresh medium (20 mM morpholineethanesulfonic acid (MES)-Tris, 145 mM NaCl; pH 5.0) with or without calcium, for 1 min at 37°C. The cells were exposed to 1 mg of pronase E/ml for 10 min at 37°C, washed with HBSS, and then were lysed with SDS lysis buffer. The samples were subjected to SDS-PAGE on a 4 to 12% gradient gel. The gels were later dried and exposed to X-ray film. To compare oligomerization-insertion of PA in the absence or presence of calcium, the corresponding bands were cut from the gels, and the associated radioactivity was measured. To study the effect of calcium channel antagonists on the oligomerization of PA, the same procedure was followed except that the cells were treated with low-pH buffer in the presence of calcium and a 100 μM concentration of the respective antagonists.

Binding of EF to receptor-bound PA. Nicked PA was produced by treating 1 μM PA with 1 μg of trypsin/ml for 45 min at 37°C. Trypsinization of PA was stopped by adding 10 μg of soybean trypsin inhibitor/ml. CHO cells plated in 24-well plates were cooled and incubated with 16 nM nicked PA in calcium-containing EMEM for 20 min at 4°C. Unbound protein was removed by washing the cells with cold calcium-free medium. Fresh medium containing radioiodinated EF (11 nM) was then added to the cells in the presence or absence of calcium. After incubation of the cells for 45 min at 4°C, they were again washed and then solubilized with 0.1 N NaOH. Radioactive counts associated with the cells were measured and were corrected for nonspecific binding to determine the specific binding of EF to receptor-bound PA. To determine the effect of various calcium-channel antagonists on the binding of EF to the receptor-bound PA, the above-mentioned procedure was followed except that the incubation of EF was allowed in the presence of 100 μM antagonist in calcium-containing medium.

Translocation of EF. CHO cells were plated in 24-well plates and were grown to confluence. The cells were washed with cold medium and were then allowed to bind to 16 nM nicked-PA in calcium-containing EMEM. After incubation of the cells for an hour at 4°C, they were again washed to remove unbound protein. CHO cells were then incubated with 11 nM radioiodinated EF for an hour at 4°C. After the cells were washed with calcium-free HBSS, they were exposed to low-pH buffer, in the presence of calcium (with or without calcium channel inhibitors) or in the absence of calcium, for 1 min at 37°C. The cells were then exposed to pronase E (1 mg/ml) for 10 min at 37°C, and then pronase inhibitors were added. The cells were washed again and then solubilized in 0.1 N NaOH. Radioactive counts associated with the cells were measured to compare translocation of EF in the presence or absence of calcium.

Delivery of EF into the cell cytosol via pinosomes. CHO cells plated in 24-well plates, were washed with hypertonic medium (HBSS medium [with or without calcium] containing 0.5 M sucrose, 10% polyethylene glycol [average Mr, 1,000], and 1% BSA), before the addition of 100 nM EF to the cells in the same medium. After incubation of the cells for 9 min at 37°C, they were washed with hypotonic medium (prepared after diluting 60 ml of HBSS medium with 40 ml of water) and then incubated at 37°C for 2.5 min. The cells were washed with calcium-free medium and incubated in medium with or without calcium. Intracellular cAMP concentrations of the cells were determined after incubation for the indicated periods of time. For studying the uptake of calcium in these cells, 45CaCl2 (5 μCi/ml) was included in the incubation medium, and the uptake of calcium by the cells was measured as described previously.

Fura-2AM-based assay for calcium influx. The Fura-2AM assay has been described in detail elsewhere (11). Briefly, the cultured cells or the cells isolated from human blood were washed with HBSS by centrifugation at 700 × g for 1 min. The cells were then resuspended in HBSS containing 1 mM CaCl2 and 1% BSA and were divided into aliquots. After incubation for 30 min, individual aliquots of the cells were treated with 3 μM Fura-2AM and 0.25 mM sulfinpyrazone (to retard transport of Fura-2AM out of the cells). Fura-2AM and sulfinpyrazone were added as 1,000-fold-concentrated stock solution in dimethyl sulfoxide, and incubation was allowed for 30 min at 37°C in dark. Extracellular dye was removed by washing the cells twice with HBSS at 700 × g for 1 min. The cells were resuspended in HBSS containing 1 or 3 mM CaCl2 and then used for the assay.

Results and Discussion

Requirement of extracellular calcium for anthrax edema toxin-induced cAMP accumulation in CHO cells. Anthrax edema toxin elicits a dose-dependent cAMP response in CHO cells. The intracellular cAMP levels of CHO cells increased more than 100-fold within 2 h of treatment with anthrax edema toxin (10 nM concentrations each of PA and EF). However, when the cells were treated with edema toxin in calcium-free medium, the toxin failed to generate cAMP response in CHO cells. In the absence of calcium, even higher doses of edema toxin (as high as 100 nM) failed to elevate intracellular cAMP levels in CHO cells (Fig. 1). The cAMP response of CHO cells to anthrax edema toxin was also abolished when the cells were treated with edema toxin in the presence of calcium chelator, EGTA (shown later as part of Fig. 3B). These results suggested that extracellular calcium was necessary for causing anthrax edema toxin-induced toxicity in CHO cells.

To understand the relationship between extracellular calcium and anthrax edema toxin-induced cAMP response of CHO cells, the cells were treated with edema toxin in calcium-free media that had been supplemented with known concentrations of calcium chloride. It was observed that the cAMP response generated by edema toxin in CHO cells was dependent on calcium concentration in the incubation medium (Fig. 2).

Identification of calcium-requiring steps during intoxication of cells by anthrax edema toxin. During intoxication of target cells by anthrax toxin, PA, the binding moiety of the toxin, binds to cell surface receptors and gets proteolytically activated to oligomerize and bind to the catalytic moieties LF and EF. It was observed that the depletion of calcium from the extracellular medium marginally affected the binding and the nicking of PA on the cell surface (Table 1). However, the oligomerization of PA or its binding to EF were not affected by the absence of calcium in the medium (Table 1).

Subsequent to the binding of the toxin to the cell surface, the toxin is taken up by the cells. The edema toxin (a 12 nM concentration of PA, along with 6 nM EF) was allowed to bind to the cell surface in calcium-containing media at 4°C. The cells were then washed with calcium-free media to remove unbound toxin and were reincubated in medium with or without calcium for 40 min, at 37°C, to allow the uptake of the toxin by the cells. The cAMP response induced by the toxin in the treated cells was then determined. It was observed that when the uptake of edema toxin was allowed in the absence of calcium, the cAMP response induced by the toxin in CHO cells was markedly reduced (76 ± 5 pmol of cAMP/mg of CHO cell protein) compared to the response generated by the toxin in the presence of extracellular calcium (704 ± 10 pmol of cAMP/mg of CHO cell protein). These results clearly demonstrate that anthrax edema toxin requires extracellular calcium at a stage subsequent to its binding.

Anthrax edema toxin bound to the cells is internalized by endocytosis. Acidification of the endosome is required for the translocation of edema factor into the cytosol. To determine whether extracellular calcium was required at a step subsequent to endocytosis of the toxin, CHO cells were preincubated with 10 mM NH4Cl and were treated with the toxin in the presence of calcium. In presence of NH4Cl, the cells take up the toxin; however, the toxin is not translocated to cytosol and remains localized in the endosome (13). After incubation of the cells for an hour, they were washed with calcium-free medium to remove calcium, unbound toxin, and NH4Cl. The cells were then incubated in medium with or without calcium. cAMP accumulation by edema factor was considerably reduced in cells that were incubated in calcium-free medium (59 ± 5 pmol of cAMP formed per mg of CHO cell protein [in 90 min]) after the removal of the amine block, in contrast to those that were incubated in calcium-containing medium (2,100 ± 20 pmol of cAMP formed per mg of CHO cell protein [in 90 min]). These results demonstrate that extracellular calcium is required for the expression of anthrax edema toxin toxicity at a stage subsequent to its internalization by endocytosis.

Acidification of the endosome causes conformational changes in PA, resulting in its insertion into the endosomal membrane, thereby forming channels for the translocation of catalytic moieties to the cytosol. The oligomerization and/or insertion of PA and the translocation of EF to the cell cytosol were unaffected by the absence of calcium in the extracellular medium (Table 1). This suggested that anthrax edema toxin required extracellular calcium at a stage subsequent to the translocation of EF into the cytosol of the target cells. To confirm this, PA-mediated delivery of EF was bypassed, and EF was directly introduced into the cytosol of CHO cells via the pinosomes. It was observed that the depletion of calcium from the extracellular medium did not affect the delivery of EF to the cell cytosol via the pinosomes (Table 2). However, the ensuing cAMP response was markedly reduced in these cells in comparison to the cells that were incubated in calcium-containing media (Table 2). These results confirmed that EF requires extracellular calcium after reaching the cell cytosol to generate an optimal cAMP response in the target cells.

Time course of 45Ca2+ uptake versus time course of cAMP accumulation. To define temporally the calcium requirement of toxin-treated cells, we measured the influx of calcium as a function of time in toxin-treated cells. A dramatic rise in the influx of calcium was recorded within 10 min of toxin treatment. Furthermore, it was observed that the intracellular cAMP levels in the toxin-treated CHO cells started rising only after 10 min of toxin treatment. Thus, on a time scale it was confirmed that the influx of calcium precedes cAMP accumulation in the toxin-treated cells (Fig. 3A). Untreated cells, or cells treated with EF alone, did not show any significant increase in influx of 45Ca2+ or any rise in the intracellular cAMP concentrations.

To study 45Ca2+ uptake in PA-treated cells, PA was allowed to bind to cell surface receptors, get proteolytically activated and oligomerize in the presence of 45Ca2+ at 4°C. It was observed that there was no increase in the uptake of 45Ca2+ in CHO cells when PA was allowed to bind to the receptors, get proteolytically activated, and oligomerize on the cell surface (Table 1). This suggests that binding, nicking, or oligomerization of PA on the cell surface does not cause the influx of calcium that occurs in toxin-treated cells. To probe further, EF was allowed to bind to receptor-bound PA at 4°C, and 45Ca2+ uptake by the cells was then measured. It was observed that the binding of EF to receptor-bound PA does not cause increased influx of calcium in the treated cells. To allow translocation of bound EF, the cells were exposed to low pH for a minute, and then 45Ca2+ uptake was measured (Table 1). It was observed that increased influx of calcium was initiated after the translocation of EF into the cell cytosol.

Similar results were obtained in another experiment in which CHO cells were treated with edema toxin in the presence of NH4Cl (to localize the toxin within the endosome). The cells were later washed (to remove amine block) and were incubated in medium supplemented with 5 μCi of 45CaCl2 (to study influx of calcium after the translocation of EF into the cell cytosol)/ml. It was observed that, 25 min after the amine block was removed, an increased influx of calcium (i.e., a 656% ± 33% increase in calcium uptake compared to the control cells) occurred in toxin-treated cells. No influx of calcium was recorded in control cells that were not treated with toxin. These results suggested that the influx of calcium in toxin-treated cells occurs only after EF reaches the cell cytosol. To further confirm this, EF was directly introduced into the cytosol (via the pinosomes). Within 10 min of EF delivery, a rapid influx of calcium occurred in the target cells (Table 2).

On the other hand, when CHO cells were treated with EF along with a translocation-defective mutant of PA, Phe427Ala (1, 28), no increase in the influx of calcium was observed (Fig. 3A). The intracellular cAMP levels also did not rise in these cells. The Phe427Ala mutant of PA binds to cell surface receptors, gets proteolytically activated, and binds to LF/EF but is unable to translocate them to the cytosol (28). Taken together, these results led us to the conclusion that influx of calcium is triggered only after the translocation of edema factor into the cytosol. Moreover, the time profiles of calcium influx and cAMP accumulation show that calcium influx precedes cAMP accumulation in the toxin treated cells.

Effect of calcium-channel antagonists on edema toxin-induced accumulation of cAMP. The observation that the influx of calcium invariably preceded the rise in cAMP levels, induced by edema toxin in the host cells, raised a pertinent question: Is influx of calcium, a prerequisite for cAMP accumulation? We reasoned that if influx of calcium necessary for edema toxin-induced cAMP accumulation, then it should be possible to inhibit cAMP production by blocking the uptake of calcium. To investigate this, we analyzed the effect of agents that block uptake of calcium on anthrax edema toxin-induced cAMP accumulation in CHO cells. The nonpermeable calcium chelator, EGTA, abolished calcium influx in toxin-treated cells and attenuated the cAMP response (Fig. 3B). La3+, which competes with calcium for several calcium channels without being transported across the plasma membrane, was able to completely prevent the entry of calcium and also the ensuing cAMP response in toxin-treated cells (Fig. 3B).

Voltage-gated channels are most typically expressed in excitable cells. However, several reports have suggested the presence of L- and T-type channels in nonexcitable cells (such as lymphocytes and fibroblasts) as well (8, 23). To investigate the involvement of these channels in mediating calcium influx in toxin-treated cells, we used the selective calcium antagonists nifedipine, verapamil, diltiazem, and flunarizine. Nifedipine, a dihydropyridine antagonist of L-type calcium channels, effectively blocked calcium influx in toxin-treated CHO cells and reduced cAMP accumulation by sixfold (Fig. 3B). However, diltiazem, a benzothiazepine antagonist of L-type calcium, did not inhibit the entry of extracellular calcium and caused a slight decrease in the cAMP response of CHO cells to edema toxin (Fig. 3B). Slight inhibition of calcium influx and cAMP response of CHO cells was observed when the cells were treated with edema toxin in presence of verapamil, a papaverine antagonist of L-type channels, whereas flunarizine, a T-type channel antagonist, effectively blocked calcium influx in CHO cells and caused significant decrease in cAMP response of CHO cells to edema toxin (Fig. 3B). When CHO cells were treated with edema toxin in presence of dandrolene, which inhibits the release of calcium from intracellular stores, it was observed that both calcium influx and cAMP accumulation were slightly affected (Fig. 3B).

Parallel studies with these calcium channel antagonists demonstrated that these inhibitors did not affect the binding, nicking, and oligomerization of PA or the binding and internalization of EF into the cytosol of these cells (Table 1). It was thus deduced that these inhibitors directly affected the calcium influx that was triggered after the translocation of EF into the host cells. The reduced level of cAMP in the toxin- and antagonist-treated cells may reflect the inability of edema factor to generate an optimal cAMP response in the presence of calcium channel blockers or may be a consequence of phosphodiesterase activity. To rule out the latter possibility, we repeated these experiments in presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX). No alteration in inhibition of cAMP production, in antagonist-treated cells, was observed (data not shown). Thus, it was deduced that the inhibition of cAMP accumulation in antagonist-treated cells was specifically because of inhibition of adenylate cyclase activity of edema factor, in these cells. Taken together, these results suggest that the influx of extracellular calcium is necessary for edema toxin induced-cAMP accumulation in the host cells.

CHO cells are quite sensitive to anthrax edema toxin. The intracellular cAMP levels of CHO cells increase as much as 200-fold upon stimulation with an optimal concentration of anthrax edema toxin. Thus, these cells provide an excellent system for the study of the biochemical events that follow toxin internalization. Having evaluated the role of extracellular calcium in inducing anthrax edema toxin-mediated cAMP response in CHO cells, we extended our study to other cell types that may serve as physiological host of edema toxin during infection.

Anthrax edema toxin requires influx of calcium for inducing cAMP toxicity in neutrophils and cultured macrophages. It has been previously demonstrated by O'Brien et al. (25) that anthrax edema toxin increases cAMP levels in neutrophils, inhibits phagocytosis, and blocks both particulate, as well as phorbol myristate acetate-induced chemiluminescence. Phagocytosis by macrophages is also inhibited when they are treated with agents that increase their intracellular cAMP concentrations (26). Several other macrophage functions, including migration, spreading, and adhesion (7), superoxide production (32), and bacterial killing are also inhibited by these agents. Thus, edema factor, by increasing intracellular cAMP concentration in macrophages and neutrophils, might be suppressing the host phagocytic response and facilitating the survival and replication of the invading organism, B. anthracis.

Both neutrophils and the cultured macrophages, RAW264.7, respond poorly to anthrax edema toxin (Fig. 4A and 5A). It was observed that extracellular calcium was necessary for causing edema toxin-induced toxicity in these cells. It was further observed that, in both the neutrophils and the cultured macrophages, treatment with anthrax edema toxin resulted in the increase in influx of extracellular calcium and that was soon followed by the rise in the intracellular cAMP levels (Fig. 4A and 5A).

To determine whether the increase in the influx of calcium essential for cAMP accumulation by edema toxin in these cells, we analyzed the cAMP response generated by edema toxin in these cells after blocking the influx of calcium. Calcium channel blockers nifedipine and diltiazem do not prevent the entry of calcium from the extracellular medium into the neutrophils, but they inhibit the movement of calcium between cytosol and the intracellular stores (27). We observed that the cAMP response generated by edema toxin in neutrophils was not affected by the presence of diltiazem or nifedipine in the medium (Fig. 4B). These results indicate that calcium release from intracellular stores has minor, if any, role in anthrax edema toxin-induced toxicity of cells. However, flunarizine, lanthium chloride, and EGTA effectively blocked calcium influx in toxin-treated neutrophils and also attenuated the cAMP response (Fig. 4B). Similarly, in toxin-treated RAW264.7 cells, the influx of calcium was suppressed by verapamil, nifedipine, diltiazem, flunarizine, and EGTA and by the competing La3+ ions (Fig. 5B). These agents also inhibited anthrax edema toxin-mediated cAMP response of these cultured macrophages (Fig. 5B). From these results we infer that anthrax edema factor requires the uptake of extracellular calcium for generating optimal cAMP response in neutrophils and cultured macrophages.

Anthrax edema toxin requires calcium influx for inducing toxicity in lymphocytes. The intracellular cAMP levels of lymphocytes undergo a striking increase after 10 min of treatment of the lymphocytes with anthrax edema toxin (Fig. 6A). The accumulation of cAMP was dose and time dependent. To the best of our knowledge, it has never been reported previously that anthrax edema toxin can cause elevation in cAMP levels of lymphocytes. Indeed, such a massive elevation in the cAMP levels of lymphocytes can lead to an alteration in of critical immunoregulatory genes (4), apoptosis (9), decrease in T-cell proliferation (29), and decrease in immune response (5). As observed with other cell types, the cAMP response generated by anthrax edema toxin in lymphocytes was dependent on extracellular calcium. cAMP levels in lymphocytes increased from basal levels of 8 ± 1 pmol/107 cells to 11 ± 2 pmol/107 cells within 1 h of treatment with anthrax edema toxin (6 nM concentrations each of PA and EF) in the absence of extracellular calcium and to 1,200 ± 45 pmol/107 cells upon treatment with the toxin in calcium-containing medium. Experiments with radioactive calcium revealed that lymphocytes respond to treatment with edema toxin by increasing the influx of calcium within the first 10 min of toxin treatment (Fig. 6A). Furthermore, it was observed that the agents that inhibited calcium uptake also downregulated cAMP accumulation by edema factor in these cells (Fig. 6B).

For a more critical evaluation of the intracellular calcium levels, at the time of the edema toxin-induced increase in calcium influx, Fura-2AM dye was used to detect changes in intracellular calcium levels in the treated lymphocytes. It was observed that the treatment of lymphocytes with PA alone did not cause any increase in the basal levels of intracellular calcium (Fig. 7). This is in agreement with our other results that led us to the conclusion that the influx of calcium that occurs in the toxin-treated cells is not caused by PA. Upon addition of EF to the PA-treated lymphocytes, a massive increase in intracellular calcium concentration, [Ca2+]i, was recorded. The elevated levels of calcium persist in the cells just for a few seconds and then rapidly decline (Fig. 7). The EF-induced increase in [Ca2+]i could be blocked by the addition of nonpermeable calcium chelator, EGTA, in the medium (data not shown). This suggested that the EF-induced increase in [Ca2+]i was caused by a potent influx of extracellular calcium in the toxin-treated cells. Further studies on Fura-2AM-loaded lymphocytes demonstrated that when lymphocytes are treated with anthrax edema toxin (12 nM PA, along with 1 nM EF) in the presence of 1 mM extracellular calcium, the EF-induced influx of calcium causes [Ca2+]i to rise 200 ± 11 nM (over the basal concentration). When the extracellular calcium concentration is increased to 3 mM, then the EF-induced influx of calcium causes [Ca2+]i to increase upto 350 ± 19 nM. This suggested that the EF-induced increase in [Ca2+]i was dependent on the extracellular calcium concentration.

The data presented here provide evidence that extracellular calcium plays a critical role in inducing anthrax edema toxin-mediated cAMP toxicity in the host cells. Depletion of calcium from the extracellular medium did not affect the PA-mediated delivery of EF into the target cells. However, after it reached the cell cytosol, EF required extracellular calcium for generating an optimal cAMP response in the host cells. It was further shown that after EF reached the cell cytosol, a rapid influx of calcium was triggered in the host cells. The surge of calcium ions was soon followed by an accumulation of cAMP in these cells. Agents that blocked calcium uptake, concomitantly inhibited edema toxin-induced accumulation of cAMP in these cells, suggesting that the influx of calcium was necessary for generating optimal cAMP response in the host cells. These calcium channel antagonists that block cAMP toxicity of anthrax edema toxin may be evaluated for their therapeutic potential against anthrax.

Several groups have shown that calcium- or calmodulin-dependent adenylate cyclases from sources as diverse as paramecium, Drosophila melanogaster, and mammalian brain (15, 33) are activated by influx of calcium. Indeed, studies on calmodulin show that transitory increase in calcium concentration was necessary for the interaction of calcium with calmodulin (17). The accompanying calcium-induced structural transitions in calmodulin coordinate the interaction of calmodulin with target enzymes and proteins, resulting in their activation (17). The fact that stimulus-response coupling mediated by calmodulin involves several steps suggests that different enzymes may be activated by different conformations of calmodulin and that stepwise changes exhibited by calmodulin at different calcium levels may be used to regulate different metabolic pathways. Thus, it is quite possible that the influx of calcium that is induced after the translocation of EF into the host cells facilitates interaction between EF and its eukaryotic activator, calmodulin. This hypothesis may also explain why the attenuation of calcium entry results in downregulation of edema toxin-mediated cAMP response.

Hitherto, most of the pathological effects associated with anthrax infection have been attributed to the lethal toxin. The contribution of the edema toxin to virulence and pathogenesis is not well understood. The data presented above show that, unlike lethal toxin, which primarily affects macrophages, edema factor affects different types of cells. By accumulating cAMP in cells of the immune system, edema factor may actually be paralyzing host immune defense, thereby facilitating replication and survival of the invading bacterium. Indeed, anthrax edema factor provides an excellent example of how cleverly B. anthracis exploits the control system that normally operates as a negative modulator of immune cell functions.

Acknowledgments

This work was partly supported by a grant from DBT (Government of India). Both P.K. and N.A. received financial assistance from UGC (Government of India).

Article - Infection and Immunity with References