Lymphoproliferative Responses to Borrelia burgdorferi in Lyme Disease David C. Zoschke, MD, PhD; Archibald A. Skemp, MD; and Dana L. Defosse, BS
Objective: To compare lymphocyte proliferative responses to Borrelia burgdorferi in healthy controls and patients with Lyme disease. Patients: Twelve patients fulfilling case-definition criteria for Lyme disease. Twelve healthy volunteers and two newborns served as controls. Measurements: Antibodies to B. burgdorferi were measured by enzyme-linked immunosorbent assay (ELISA). Proliferation of peripheral blood lymphocytes cultured for 5 days with B. burgdorferi, recall antigens, or pokeweed mitogen was measured by radioactive thymidine uptake. Results: Lymphocytes from 11 patients with Lyme disease, 8 healthy seronegative controls, and two newborns showed elevated responses when stimulated with B. burgdorferi. When a patient and a control were studied on the same day, the patient's lymphocyte response to B. burgdorferi exceeded the control's in only 5 of 12 cases. Lymphocytes from both patients and controls responded to B. burgdorferi isolates from three different sources. Conclusions: Heightened lymphocyte responses to B. burgdorferi are found in patients with Lyme disease but elevated responses also frequently occur in healthy controls. At present, the interpretation of a positive lymphocyte response to B. burgdorferi would be difficult in ambiguous clinical situations. Annals of Internal Medicine. 1991;114:285-289. From the University of Minnesota, Minneapolis, Minnesota. For current author addresses, see the end of text.
L y m e borreliosis is a potentially chronic infection caused by Borrelia burgdorferi, a tick-borne spirochete. The infection is characterized initially by a distinct rash, erythema migrans; cardiac, neurologic, and arthritic features occur later and are accompanied by a specific antibody response to B. burgdorferi. In a recent study, 17 patients with antibiotic-treated erythema migrans subsequently developed symptoms and findings associated with late disease (1). Despite an absence of antibodies to B. burgdorferi, these patients showed heightened in-vitro lymphocyte proliferative responses to B. burgdorferi, confirming previous exposure. Awareness of and concern about Lyme disease is increasing, especially in endemic areas. Several aspects concerning diagnosis are particularly disconcerting. First, some patients do not recall having or do not experience erythema migrans, which is still the best clinical marker. Second, it appears that some patients with late disease may not have detectable serologic
responses (1). Indeed, it is a common conviction among prospective patients that the antibody test is insensitive and unreliable. Because of the widespread publicity about the symptoms associated with Lyme disease, there has been a dramatic increase in clinic visits by persons who have not had either a tick bite or a rash; these patients have only low levels of antibodies to B. burgdorferi and no disease findings, but they do have chronic fatigue associated with vague neurologic and rheumatic symptoms. Some have received prolonged antibiotic therapy with varying results, but only rarely do they experience complete symptom remission. The potential value of a lymphoproliferative assay for B. burgdorferi infection in such situations is apparent. If such an assay is more sensitive than serologic testing, then a negative response should rule out significant exposure. We began testing lymphocytes from healthy controls and patients with Lyme disease, establishing their response patterns to ascertain the potential usefulness of the assay in ambiguous clinical situations. We found that although lymphocytes from patients with Lyme disease respond in vitro to B. burgdorferi, a similar reactivity is often found with lymphocytes from healthy controls. Methods Mononuclear leukocytes were obtained from 12 patients meeting the criteria for Lyme disease and from cord bloods of 2 healthy newborns. Diagnosis was based on exposure in an endemic area and clinical findings, including either documented erythema migrans or seropositivity combined with characteristic rheumatologic, neurologic, or cardiac findings (2). Each patient was tested on a different day. Because of the inherent day-to-day variation in the results of lymphocyte cultures, leukocytes were also prepared and tested each day from a different healthy volunteer. No attempt was made to match characteristics between patients and healthy controls. All controls were seronegative for B. burgdorferi antibodies. Peripheral venous blood was drawn into heparinized syringes, and mononuclear cells were prepared as previously described (3). Briefly, the cellular fraction was diluted with Hanks balanced salt solution and incubated with 6% hetastarch in 0.9% sodium chloride; the leukocyte-rich supernatant was further separated on one-step discontinuous Percoll gradients (Pharmacia, Piscataway, New Jersey). Mononuclear leukocytes were harvested from the supernatant-1.076 density interface and gently washed twice with Hanks balanced salt solution. Cells were resuspended in culture medium consisting of RPMI 1640 (Gibco, Grand Island, New York), 0.02M Hepes buffer, 100 U/mL penicillin-streptomycin (Sigma, St. Louis, Missouri), and 15% normal human heat-inactivated plasma. Cells were cultured at 1 x 105 cells per well in flat-bottomed microculture plates in a final volume of 225 /til. B. burgdorferi organisms, specific recall antigens, or pokeweed mitogen (Gibco) were added in 25 /A to triplicate cultures. The recall antigen preparations, Candida albicans (Hollister-Stier, Spokane, Washington) and tetanus toxoid (Connaught, Swiftwater, Pennsylvania), were dialyzed overnight against Hanks balanced salt solution to remove inhibitory preservatives and used ©1991 American College of Physicians
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Table 1. Clinical Characteristics and Lymphocyte Lyme Disease and Healthy Controls Patient 1
Patients with Lyme Disease ELISA B. burgdorferi Value* Response
Cranial nerve palsy; arthritis Heart block; arthritis Erythema migrans Arthritis Arthritis Erythema migrans Tick bite; arthritis Arthritis Tick bite; cranial nerve palsy Tick bite; arthritis Tick bite; erythema migrans; arthritis Tick bite, erythema migrans
2 3 4 5 6 7 8 9 10 11 12 Mean * t $ §
Clinical Findings
Proliferative Responses
Background Responset
Stimulation Index
to Borrelia burgdorferi in Patients
with
Healthy Controls B. burgdorferi Background Stimulation Response Response! Index
0.65 1.49 1.08 0.49 1.63 0.29 0.14 0.94
7 313$ 22 948$§ 6 253$§ 8 368$§ 14 131$§ 7 461$ 1830$ 2 101
826 212 662 254 1 419 83 138 1 101
8.9 108.2 9.5 32.9 9.9 89.9 13.3 1.9
4 501$ 5 515$ 2 901$ 2 128$ 131 3 510$ 495 1 395
762 312 1120 34 98 301 34 1 281
5.9 17.7 2.6 62.6 1.3 11.6 14.6 1.1
0.54 0.90
11 045$§ 1735$
987 162
11.2 10.7
4 126$ 1110$
984 49
4.2 22.7
0.34
12 438
1124
11.1
11427$
1016
11.2
0.49
13 170$ 9 066$§
626 638
21.0 27.4
11906$ 4 095$
962 579
12.4 13.9
ELISA = enzyme-linked immunosorbent assay at the time of lymphocyte testing. Mean background count/min of unstimulated cultures. Mean B. burgdorferi response was greater than mean background response (P < 0.05). Mean B. burgdorferi response in the patient was greater than that seen in the healthy control tested on the same day {P < 0.05).
at near optimal concentrations predetermined in several normal persons. Poke weed mitogen was used at a final concentration of 1:250 and served as a positive culture control to ensure the presence of potentially responsive lymphocytes in both patient and control cell preparations. Cultures were maintained for 136 hours at 37 °C in a humidified atmosphere containing 5% carbon dioxide and then pulsed for 6 hours with 0.5 /iCi tritiated thymidine (specific activity, 6.7 Ci/mmol, New England Nuclear, Boston, Massachusetts). Cultures were harvested with a semi-automated collector (Cambridge Technology, Inc., Watertown, Massachusetts), and tritiated thymidine incorporation was determined by liquid scintillation spectroscopy. Three different B. burgdorferi isolates were grown and passaged in modified Kelly medium (4). Connecticut isolate 297 was originally obtained from human cerebospinal fluid, Federal Republic of Germany isolate P/GU from an erythema migrans lesion, and New York isolate B31 from Ixodes dammini (ATCC 35210). After 5 to 7 days of growth, spirochetes were collected by centrifugation at 12 000 g for 20 min at 4 °C. After the spirochetes were washed three times in Hanks salt solution, they were resuspended in RPMI 1640, enumerated using dark-field microscopy, and added to cell cultures at concentrations ranging from 1 x 102 to 1 x 107 organisms per culture. Data presented are for concentrations of 1 x 106 organisms, which usually gave the optimum response. Borrelia burgdorferi cultured alone in cell media did not incorporate tritiated thymidine. An enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin to B. burgdorferi was done on serum specimens from each patient and control. The method used was identical to that of Magnarelli and colleagues (5) except that the antigen was sonicated B. burgdorferi isolate 297. Based on values obtained from study of more than 600 serum specimens from normal subjects, optical density values of 0.886 or greater are 3 standard deviations above the mean and are considered indicative of significant B. burgdorferi antibody levels. Experimental data are presented as the mean (±SE) count per minute (cpm) of triplicate cultures and as stimulation indices (mean cpm with stimulant/mean cpm without stimulant). The statistical significance of differences between mean values was analyzed with the Student two-tailed paired /-test. When appropriate, the 95% confidence interval (CI) is given. 286
Results Clinical Manifestations The clinical characteristics of the 12 patients with Lyme disease are shown in Table 1. All patients resided in or frequently visited areas of east central Minnesota endemic for B. burgdorferi-infected Ixodes dammini. Several patients recalled a specific tick bite. Constitutional symptoms such as fatigue, headache, and myalgia were universal. Borrelia burgdorferi antibody levels at the time of lymphocyte testing ranged from negative to strongly positive. However, all patients had elevated B. burgdorferi antibody levels when diagnosed except for Patients 6 and 12, who received antibiotic therapy when erythema migrans was recognized. Lymphocyte Proliferative Response to Borrelia burgdorferi The mean lymphocyte responses to B. burgdorferi isolate 297 are shown in Table 1. Except for Patient 8, patients with Lyme disease showed elevated B. burgdorfe ri-induced responses (P < 0.05) when compared with background proliferation. However, 8 of the 12 healthy controls also had elevated responses that were statistically significant (P < 0.05). Test sensitivity (11 of 12 patients positive) was 92% (CI, 62% to 100%), and test specificity (4 of 12 healthy controls negative) was 33% (CI, 10% to 65%). When patient and control were tested on the same day, the response of the patient exceeded that of the healthy control in only 5 of 12 cases. The overall mean (±SE) B. burgdorferi-induccd response of patients with Lyme disease (9066 ± 1782 cpm) was higher than that of the healthy controls (4095 ± 1126 cpm; P = 0.009, by unpaired Mest). Stimulation indices ranged widely, in part because of varia-
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tions in background proliferation. The mean stimulation index of patients did not differ from that of controls (27.4 ± 9.9 compared with 13.9 ± 4.8; P = 0.2). To determine if B. burgdorferi isolate 297 might have unique mitogen-like properties, two other B. burgdorferi isolates, the ATCC standard, B31, and isolate P/GU, were tested with cells from five healthy controls prepared the same day. All five healthy controls responded to each different B. burgdorferi isolate in a dose-dependent manner (Figure 1). The mean maximum stimulation index ranged between 9 and 13. Responses to the different B. burgdorferi isolates were similar to those found with Candida albicans and tetanus recall antigens. Our healthy controls also resided in or visited areas considered endemic for B. burgdorferi-infected Ixodes dammini. Thus, despite uniformly negative serologic tests, they could have been unknowingly exposed and sensitized to B. burgdorferi. Therefore, cord-blood cells from two healthy newborns delivered by healthy seronegative mothers were also tested (Figure 2). The cord-blood lymphocytes and the adult control cells responded both to B. burgdorferi and to poke weed mitogen. As expected, these immunologically naive lymphocytes did not respond to Candida albicans recall antigen whereas the adult lymphocytes did respond.
Discussion In measuring in-vitro lymphocyte proliferative responses to B. burgdorferi, our most important observation concerned the statistically elevated responses occurring in 8 of 12 lymphocyte samples from seronegative healthy controls and in both cord-blood samples. In fact, when a patient's response was compared with that of a healthy control tested the same day, in only 5 of 12 cases did the response of the patient statistically exceed that of the control (P < 0.05). Further, lymphocytes from healthy controls responded to three different B. burgdorferi isolates, and responses were quantitatively similar to those seen with classic recall antigens. Taken together, our results indicate that part of the lymphocyte response induced by B. burgdorferi does not require previous antigen exposure. All patients fulfilled case-definition criteria for Lyme disease, and some had completed antibiotic therapy before the lymphocyte proliferation assay. Nevertheless, active disease or a currently positive serologic test did not necessarily predict a strong response to B. burgdorferi. Indeed, the most vigorous response was seen in a patient who had had successful antibiotic treatment 1 year previously (Patient 2), whereas the weakest response occurred in a seropositive patient with active arthritis (Patient 8). The number of patients in our study was small, although many potential subjects had been seen in clinic. Some had been treated elsewhere for Lyme disease, with diagnosis based only on residence in an endemic area and subjective physical ailments. Others were seropositive, but extensive diagnostic testing did not show rheumatologic, neurologic, or cardiac disease. We intentionally avoided testing the former group, but several in the latter group were tested. Their
responses to B. burgdorferi also varied, with the stimulation index ranging from 2.7 to 7.9. The current, albeit limited, information on the clinical and immunologic features of chronic Lyme disease has recently been reviewed (2, 6). A B. burgdorferi-specific immune response occurs during infection. Because B. burgdorferi is not readily cultured or demonstrated in tissue specimens, detection of this immune response is a crucial diagnostic aid. The ELISA has become the preferred test for detecting B. burgdorferi antibodies, although serious interlaboratory and intralaboratory variability exists (7). Cellular immune responses to B. burgdorferi have been examined in laboratory animals and in humans. Benach and colleagues (8) found impressive in-vitro proliferative responses in B. burgdorferi-stimulated splenic lymphocytes from both control and infected BALB/c mice. In contrast, Schaible and colleagues (9) reported that after Borrelia inoculation, BALB/c mice developed weaker delayed-type hypersensitivity responses compared with other sensitized inbred murine strains. Lymphocytes from these sensitized strains of mice, such as C57BL/6, had a fivefold greater proliferative response to a B. burgdorferi-sohible antigen preparation than did their unexposed litter mates. Sigal and colleagues (10) initially reported that lymphocytes from certain human subjects responded to a sonicated B. burgdorferi preparation. Patients with Lyme disease who had active arthritis had the strongest responses, although responses tended to decrease after antibiotic treatment. Mononuclear cells from synovial fluid responded more vigorously than did blood mononuclear cells, suggesting localization of immune activity. Low responses were reported in unrelated healthy controls. Although our methods differed from those
Figure 1. The mean lymphocyte proliferative responses of five healthy controls to three different Borrelia burgdorferi (Bb) isolates and to Candida and tetanus recall antigens. Responses are presented as the mean (±SE) stimulation index.
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Figure 2. The proliferative responses of umbilical cord blood lymphocytes from newborns and lymphocytes from healthy adults to poke weed mitogen, Candida recall antigen, and Borrelia burgdorferi. (A CPM = the mean counts per minute [CPM] of triplicate cultures with stimulant added minus the mean cpm [triplicate] of background thymidine incorporation; Nl = normal.)
used by Sigal and colleagues (10), we found that a strong lymphocyte response may persist long after successful treatment. Freshly prepared B. burgdorferi organisms co-cultured with lymphocytes at 1:1 to 10:1 ratios generally induce optimal lymphocyte responses. Dattwyler and colleagues (1) applied this method to patients who were designated as having Lyme disease but were seronegative for B. burgdorferi antibodies. This diagnosis was based on previous erythema migrans, flu symptoms, residence in an endemic area, and low B. burgdorferi antibody levels in the setting of late disease. The mean response to B. burgdorferi in these patients was similar to that seen in seropositive patients with Lyme disease and was significantly greater than that seen in the healthy control group (P < 0.05). However, overlap with controls was seen within both patient groups. The apparent dissociation between humoral and cellular immunity was thought to be due to inadequate early anti288
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biotic treatment. Presumably, a reduction in antigen levels aborted maturation of a humoral response but not before the infection disseminated and T cells became sensitized. Our results confirm that, as a group, patients with Lyme disease have higher mean lymphocyte responses to B. burgdorferi than do healthy controls. Unfortunately, healthy controls also showed lymphocyte responses to B. burgdorferi and, in most cases, lymphocyte proliferation testing could not discriminate between the patient and the healthy control tested that same day. Lymphocyte stimulation by B. burgdorferi appears to have both specific and nonspecific features. The response to B. burgdorferi seen in immunologically naive fetal cord-blood lymphocytes suggests a nonspecific mitogen-like response. Sigal and colleagues (11) recently noted similar nonspecificity; in their study, lymphocytes from healthy controls produced increased IgM in vitro after B. burgdorferi stimulation. Increased antinuclear antibodies and rheumatoid factor rates reported in patients with Lyme disease may reflect polyclonal B-cell activation (12). A lipopolysaccharide isolated from B. burgdorferi has been reported to stimulate normal lymphocyte proliferation (13). Normal human monocytes release considerable interleukin-1 when exposed to B. burgdorferi, and lipopoly saccharide depletion by polymyxin does not affect this response (14). Once present, interleukin-1 could considerably augment other minor mitogenic stimulants. Our observation that the overall mean lymphocyte response seen in patients was significantly higher than the mean response seen in controls may indicate some degree of immune specificity. In addition, the individual responses seen in controls never exceeded those seen in patients. Efforts to improve this test's specificity by fractionating B. burgdorferi antigens are in progress (15, 16). The assay could also possibly be improved by simultaneously testing lymphocytes from a large panel of controls and patients known to have Lyme disease. Lymphocytes from patients might be preserved for culturing using cryopreservation technology. We hoped that lymphocyte testing might prove to be useful in ruling out exposure to B. burgdorferi. Based on our experience, lymphocyte proliferation induced by B. burgdorferi is possible regardless of exposure status, and a positive response by itself has little clinical significance. Clearly, interactions between B. burgdorferi and the immune system occur. The resultant immune response may be crucial in either resolving the infection or initiating pathologic changes with prolonged clinical manifestations (17-19). Until such studies are completed, however, we believe that lymphocyte response testing has little to add in either the diagnosis or the management of Lyme disease. Acknowledgments: The authors thank Dr. Russell Johnson, Department of Microbiology, University of Minnesota, for doing the ELISAs for B. burgdorferi and for providing the B. burgdorferi isolates. They also thank Leslie Deem and Joanne Bailey for secretarial assistance. Grant Support: In part by National Institutes of Health grant 5R01-AR33492. Requests for Reprints: David C. Zoschke, MD, Department of Medicine, Section of Rheumatology, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455.
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Current Author Addresses: Drs. Zoschke and Skemp and Mr. Defosse: Department of Medicine, Section of Rheumatology, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455. References 1. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golightly MG. Seronegative lyme disease: dissociation of the specific T- and B-lymphocyte responses to Borrelia burgdorferi. N Engl J Med. 1988;319:1441-6. 2. Steere AC. Lyme Disease. N Engl J Med. 1989;321:586-96. 3. Zoschke DC, Kaja J. Suboptimal levels of hydrogen peroxide scavengers in synovial fluid: in vitro augmentation with slow acting anti-rheumatic drugs. J Rheum. 1989;16:1233-40. 4. Steere AC, Grodzicki AN, Kornblatt AN, et al. The spirochetal etiology of Lyme disease. N Engl J Med. 1983;308:733-40. 5. Magnarelli LA, Anderson JF. Enzyme-linked immunosorbent assays for the detection of class-specific immunoglobulins to Borrelia burgdorferi. Am J Epidemiol. 1988;127:818-25. 6. Sigal LH. Lyme disease, 1988: immunologic manifestations and possible immunopathogenetic mechanisms. Semin Arthritis Rheum. 1989;18:151-67. 7. Schwartz BS, Goldstein MD, Ribeiro JMC, Schulze TL, Shahied SI. Antibody testing in Lyme disease. A comparison of results in four laboratories. JAMA. 1989;262:3431-4. 8. Benach JL, Coleman JL, Garcia-Monco JC, Deponte PC. Biological activity of Bb antigens. Ann NY Acad of Sci. 1988;539:115-25. 9. Schaible UE, Kramer MD, Justus CW, Museteneau C, Simon MM. Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi. Infect Immun. 1989;57:41-47.
10. Sigal LH, Steere AC, Freeman DH, et al. Proliferative response of mononuclear cells in Lyme disease. Arthritis Rheum. 1986;29:761-9. 11. Sigal LH, Steere AC, Dwyer JM. In vivo and in vitro evidence of B cell hyperactivity during Lyme disease. J Rheumatol. 1988;15:64854. 12. Goebel KM, Krause A, Neurath F. Acquired transient autoimmune reactions in Lyme arthritis: correlation between rheumatoid factor and disease activity. Scand J Rheum. 1988;75(Supp):314-7. 13. Beck G, Habicht GS, Benach JL, Coleman JL. Chemical and biologic characterization of a lipopolysaccharide extracted from the Lyme disease spirochete (Borrelia burgdorferi). J Infect Dis. 1985; 152:108-17. 14. Habicht GS, Beck G, Benach JL, Coleman JL, Leichtling KD. Lyme disease spirochetes induce human and murine interleukin-1 production. J Immunol. 1985;134:3147-54. 15. Yoshinari NH, Reinhardt BN, Steere AC. Components of B. burgdorferi causing T-cell proliferative responses in patients with Lyme arthritis [Abstract]. Arthritis Rheum. 1989;546:31. 16. Dattwyler R, Volkman P, Shepard D, Gorevic P. Characterization of the cell immune response to individual Borrelia burgdorferi antigens in Lyme borreliosis [Abstract]. Arthritis Rheum. 1989;80(Suppl):52. 17. Martin R, Ortlauf J, Sticht-Groh V, Bogjahn V, Goldman SF, Mertens HG. Borrelia burgdorferi-specific and autoreactive T-cell lines from cerebrospinal fluid in Lyme radiculomyelitis. Ann Neurol. 1988;24:509-16. 18. Sigal LH, Tatum AH. Tissue-specific auto-antibodies in a Lyme disease neurologic patient's sera bind to cross reacting B. burgdorferi and neuronal antigens. Arthritis Rheum. 1987;30:536. 19. Weyand LM, Goronzy JJ. Immune responses to Borrelia burgdorferi in patients with reactive arthritis. Arthritis Rheum. 1980;32:1057-64.
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Objective: To compare lymphocyte proliferative responses to Borrelia burgdorferi in healthy controls and patients with Lyme disease. Patients: Twelve patients fulfilling case-definition criteria for Lyme disease. Twelve healthy volunteers and two newborns served as controls. Measurements: Antibodies to B. burgdorferi were measured by enzyme-linked immunosorbent assay (ELISA). Proliferation of peripheral blood lymphocytes cultured for 5 days with B. burgdorferi, recall antigens, or pokeweed mitogen was measured by radioactive thymidine uptake. Results: Lymphocytes from 11 patients with Lyme disease, 8 healthy seronegative controls, and two newborns showed elevated responses when stimulated with B. burgdorferi. When a patient and a control were studied on the same day, the patient's lymphocyte response to B. burgdorferi exceeded the control's in only 5 of 12 cases. Lymphocytes from both patients and controls responded to B. burgdorferi isolates from three different sources. Conclusions: Heightened lymphocyte responses to B. burgdorferi are found in patients with Lyme disease but elevated responses also frequently occur in healthy controls. At present, the interpretation of a positive lymphocyte response to B. burgdorferi would be difficult in ambiguous clinical situations. Annals of Internal Medicine. 1991;114:285-289. From the University of Minnesota, Minneapolis, Minnesota. For current author addresses, see the end of text.
L y m e borreliosis is a potentially chronic infection caused by Borrelia burgdorferi, a tick-borne spirochete. The infection is characterized initially by a distinct rash, erythema migrans; cardiac, neurologic, and arthritic features occur later and are accompanied by a specific antibody response to B. burgdorferi. In a recent study, 17 patients with antibiotic-treated erythema migrans subsequently developed symptoms and findings associated with late disease (1). Despite an absence of antibodies to B. burgdorferi, these patients showed heightened in-vitro lymphocyte proliferative responses to B. burgdorferi, confirming previous exposure. Awareness of and concern about Lyme disease is increasing, especially in endemic areas. Several aspects concerning diagnosis are particularly disconcerting. First, some patients do not recall having or do not experience erythema migrans, which is still the best clinical marker. Second, it appears that some patients with late disease may not have detectable serologic
responses (1). Indeed, it is a common conviction among prospective patients that the antibody test is insensitive and unreliable. Because of the widespread publicity about the symptoms associated with Lyme disease, there has been a dramatic increase in clinic visits by persons who have not had either a tick bite or a rash; these patients have only low levels of antibodies to B. burgdorferi and no disease findings, but they do have chronic fatigue associated with vague neurologic and rheumatic symptoms. Some have received prolonged antibiotic therapy with varying results, but only rarely do they experience complete symptom remission. The potential value of a lymphoproliferative assay for B. burgdorferi infection in such situations is apparent. If such an assay is more sensitive than serologic testing, then a negative response should rule out significant exposure. We began testing lymphocytes from healthy controls and patients with Lyme disease, establishing their response patterns to ascertain the potential usefulness of the assay in ambiguous clinical situations. We found that although lymphocytes from patients with Lyme disease respond in vitro to B. burgdorferi, a similar reactivity is often found with lymphocytes from healthy controls. Methods Mononuclear leukocytes were obtained from 12 patients meeting the criteria for Lyme disease and from cord bloods of 2 healthy newborns. Diagnosis was based on exposure in an endemic area and clinical findings, including either documented erythema migrans or seropositivity combined with characteristic rheumatologic, neurologic, or cardiac findings (2). Each patient was tested on a different day. Because of the inherent day-to-day variation in the results of lymphocyte cultures, leukocytes were also prepared and tested each day from a different healthy volunteer. No attempt was made to match characteristics between patients and healthy controls. All controls were seronegative for B. burgdorferi antibodies. Peripheral venous blood was drawn into heparinized syringes, and mononuclear cells were prepared as previously described (3). Briefly, the cellular fraction was diluted with Hanks balanced salt solution and incubated with 6% hetastarch in 0.9% sodium chloride; the leukocyte-rich supernatant was further separated on one-step discontinuous Percoll gradients (Pharmacia, Piscataway, New Jersey). Mononuclear leukocytes were harvested from the supernatant-1.076 density interface and gently washed twice with Hanks balanced salt solution. Cells were resuspended in culture medium consisting of RPMI 1640 (Gibco, Grand Island, New York), 0.02M Hepes buffer, 100 U/mL penicillin-streptomycin (Sigma, St. Louis, Missouri), and 15% normal human heat-inactivated plasma. Cells were cultured at 1 x 105 cells per well in flat-bottomed microculture plates in a final volume of 225 /til. B. burgdorferi organisms, specific recall antigens, or pokeweed mitogen (Gibco) were added in 25 /A to triplicate cultures. The recall antigen preparations, Candida albicans (Hollister-Stier, Spokane, Washington) and tetanus toxoid (Connaught, Swiftwater, Pennsylvania), were dialyzed overnight against Hanks balanced salt solution to remove inhibitory preservatives and used ©1991 American College of Physicians
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Table 1. Clinical Characteristics and Lymphocyte Lyme Disease and Healthy Controls Patient 1
Patients with Lyme Disease ELISA B. burgdorferi Value* Response
Cranial nerve palsy; arthritis Heart block; arthritis Erythema migrans Arthritis Arthritis Erythema migrans Tick bite; arthritis Arthritis Tick bite; cranial nerve palsy Tick bite; arthritis Tick bite; erythema migrans; arthritis Tick bite, erythema migrans
2 3 4 5 6 7 8 9 10 11 12 Mean * t $ §
Clinical Findings
Proliferative Responses
Background Responset
Stimulation Index
to Borrelia burgdorferi in Patients
with
Healthy Controls B. burgdorferi Background Stimulation Response Response! Index
0.65 1.49 1.08 0.49 1.63 0.29 0.14 0.94
7 313$ 22 948$§ 6 253$§ 8 368$§ 14 131$§ 7 461$ 1830$ 2 101
826 212 662 254 1 419 83 138 1 101
8.9 108.2 9.5 32.9 9.9 89.9 13.3 1.9
4 501$ 5 515$ 2 901$ 2 128$ 131 3 510$ 495 1 395
762 312 1120 34 98 301 34 1 281
5.9 17.7 2.6 62.6 1.3 11.6 14.6 1.1
0.54 0.90
11 045$§ 1735$
987 162
11.2 10.7
4 126$ 1110$
984 49
4.2 22.7
0.34
12 438
1124
11.1
11427$
1016
11.2
0.49
13 170$ 9 066$§
626 638
21.0 27.4
11906$ 4 095$
962 579
12.4 13.9
ELISA = enzyme-linked immunosorbent assay at the time of lymphocyte testing. Mean background count/min of unstimulated cultures. Mean B. burgdorferi response was greater than mean background response (P < 0.05). Mean B. burgdorferi response in the patient was greater than that seen in the healthy control tested on the same day {P < 0.05).
at near optimal concentrations predetermined in several normal persons. Poke weed mitogen was used at a final concentration of 1:250 and served as a positive culture control to ensure the presence of potentially responsive lymphocytes in both patient and control cell preparations. Cultures were maintained for 136 hours at 37 °C in a humidified atmosphere containing 5% carbon dioxide and then pulsed for 6 hours with 0.5 /iCi tritiated thymidine (specific activity, 6.7 Ci/mmol, New England Nuclear, Boston, Massachusetts). Cultures were harvested with a semi-automated collector (Cambridge Technology, Inc., Watertown, Massachusetts), and tritiated thymidine incorporation was determined by liquid scintillation spectroscopy. Three different B. burgdorferi isolates were grown and passaged in modified Kelly medium (4). Connecticut isolate 297 was originally obtained from human cerebospinal fluid, Federal Republic of Germany isolate P/GU from an erythema migrans lesion, and New York isolate B31 from Ixodes dammini (ATCC 35210). After 5 to 7 days of growth, spirochetes were collected by centrifugation at 12 000 g for 20 min at 4 °C. After the spirochetes were washed three times in Hanks salt solution, they were resuspended in RPMI 1640, enumerated using dark-field microscopy, and added to cell cultures at concentrations ranging from 1 x 102 to 1 x 107 organisms per culture. Data presented are for concentrations of 1 x 106 organisms, which usually gave the optimum response. Borrelia burgdorferi cultured alone in cell media did not incorporate tritiated thymidine. An enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin to B. burgdorferi was done on serum specimens from each patient and control. The method used was identical to that of Magnarelli and colleagues (5) except that the antigen was sonicated B. burgdorferi isolate 297. Based on values obtained from study of more than 600 serum specimens from normal subjects, optical density values of 0.886 or greater are 3 standard deviations above the mean and are considered indicative of significant B. burgdorferi antibody levels. Experimental data are presented as the mean (±SE) count per minute (cpm) of triplicate cultures and as stimulation indices (mean cpm with stimulant/mean cpm without stimulant). The statistical significance of differences between mean values was analyzed with the Student two-tailed paired /-test. When appropriate, the 95% confidence interval (CI) is given. 286
Results Clinical Manifestations The clinical characteristics of the 12 patients with Lyme disease are shown in Table 1. All patients resided in or frequently visited areas of east central Minnesota endemic for B. burgdorferi-infected Ixodes dammini. Several patients recalled a specific tick bite. Constitutional symptoms such as fatigue, headache, and myalgia were universal. Borrelia burgdorferi antibody levels at the time of lymphocyte testing ranged from negative to strongly positive. However, all patients had elevated B. burgdorferi antibody levels when diagnosed except for Patients 6 and 12, who received antibiotic therapy when erythema migrans was recognized. Lymphocyte Proliferative Response to Borrelia burgdorferi The mean lymphocyte responses to B. burgdorferi isolate 297 are shown in Table 1. Except for Patient 8, patients with Lyme disease showed elevated B. burgdorfe ri-induced responses (P < 0.05) when compared with background proliferation. However, 8 of the 12 healthy controls also had elevated responses that were statistically significant (P < 0.05). Test sensitivity (11 of 12 patients positive) was 92% (CI, 62% to 100%), and test specificity (4 of 12 healthy controls negative) was 33% (CI, 10% to 65%). When patient and control were tested on the same day, the response of the patient exceeded that of the healthy control in only 5 of 12 cases. The overall mean (±SE) B. burgdorferi-induccd response of patients with Lyme disease (9066 ± 1782 cpm) was higher than that of the healthy controls (4095 ± 1126 cpm; P = 0.009, by unpaired Mest). Stimulation indices ranged widely, in part because of varia-
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tions in background proliferation. The mean stimulation index of patients did not differ from that of controls (27.4 ± 9.9 compared with 13.9 ± 4.8; P = 0.2). To determine if B. burgdorferi isolate 297 might have unique mitogen-like properties, two other B. burgdorferi isolates, the ATCC standard, B31, and isolate P/GU, were tested with cells from five healthy controls prepared the same day. All five healthy controls responded to each different B. burgdorferi isolate in a dose-dependent manner (Figure 1). The mean maximum stimulation index ranged between 9 and 13. Responses to the different B. burgdorferi isolates were similar to those found with Candida albicans and tetanus recall antigens. Our healthy controls also resided in or visited areas considered endemic for B. burgdorferi-infected Ixodes dammini. Thus, despite uniformly negative serologic tests, they could have been unknowingly exposed and sensitized to B. burgdorferi. Therefore, cord-blood cells from two healthy newborns delivered by healthy seronegative mothers were also tested (Figure 2). The cord-blood lymphocytes and the adult control cells responded both to B. burgdorferi and to poke weed mitogen. As expected, these immunologically naive lymphocytes did not respond to Candida albicans recall antigen whereas the adult lymphocytes did respond.
Discussion In measuring in-vitro lymphocyte proliferative responses to B. burgdorferi, our most important observation concerned the statistically elevated responses occurring in 8 of 12 lymphocyte samples from seronegative healthy controls and in both cord-blood samples. In fact, when a patient's response was compared with that of a healthy control tested the same day, in only 5 of 12 cases did the response of the patient statistically exceed that of the control (P < 0.05). Further, lymphocytes from healthy controls responded to three different B. burgdorferi isolates, and responses were quantitatively similar to those seen with classic recall antigens. Taken together, our results indicate that part of the lymphocyte response induced by B. burgdorferi does not require previous antigen exposure. All patients fulfilled case-definition criteria for Lyme disease, and some had completed antibiotic therapy before the lymphocyte proliferation assay. Nevertheless, active disease or a currently positive serologic test did not necessarily predict a strong response to B. burgdorferi. Indeed, the most vigorous response was seen in a patient who had had successful antibiotic treatment 1 year previously (Patient 2), whereas the weakest response occurred in a seropositive patient with active arthritis (Patient 8). The number of patients in our study was small, although many potential subjects had been seen in clinic. Some had been treated elsewhere for Lyme disease, with diagnosis based only on residence in an endemic area and subjective physical ailments. Others were seropositive, but extensive diagnostic testing did not show rheumatologic, neurologic, or cardiac disease. We intentionally avoided testing the former group, but several in the latter group were tested. Their
responses to B. burgdorferi also varied, with the stimulation index ranging from 2.7 to 7.9. The current, albeit limited, information on the clinical and immunologic features of chronic Lyme disease has recently been reviewed (2, 6). A B. burgdorferi-specific immune response occurs during infection. Because B. burgdorferi is not readily cultured or demonstrated in tissue specimens, detection of this immune response is a crucial diagnostic aid. The ELISA has become the preferred test for detecting B. burgdorferi antibodies, although serious interlaboratory and intralaboratory variability exists (7). Cellular immune responses to B. burgdorferi have been examined in laboratory animals and in humans. Benach and colleagues (8) found impressive in-vitro proliferative responses in B. burgdorferi-stimulated splenic lymphocytes from both control and infected BALB/c mice. In contrast, Schaible and colleagues (9) reported that after Borrelia inoculation, BALB/c mice developed weaker delayed-type hypersensitivity responses compared with other sensitized inbred murine strains. Lymphocytes from these sensitized strains of mice, such as C57BL/6, had a fivefold greater proliferative response to a B. burgdorferi-sohible antigen preparation than did their unexposed litter mates. Sigal and colleagues (10) initially reported that lymphocytes from certain human subjects responded to a sonicated B. burgdorferi preparation. Patients with Lyme disease who had active arthritis had the strongest responses, although responses tended to decrease after antibiotic treatment. Mononuclear cells from synovial fluid responded more vigorously than did blood mononuclear cells, suggesting localization of immune activity. Low responses were reported in unrelated healthy controls. Although our methods differed from those
Figure 1. The mean lymphocyte proliferative responses of five healthy controls to three different Borrelia burgdorferi (Bb) isolates and to Candida and tetanus recall antigens. Responses are presented as the mean (±SE) stimulation index.
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Figure 2. The proliferative responses of umbilical cord blood lymphocytes from newborns and lymphocytes from healthy adults to poke weed mitogen, Candida recall antigen, and Borrelia burgdorferi. (A CPM = the mean counts per minute [CPM] of triplicate cultures with stimulant added minus the mean cpm [triplicate] of background thymidine incorporation; Nl = normal.)
used by Sigal and colleagues (10), we found that a strong lymphocyte response may persist long after successful treatment. Freshly prepared B. burgdorferi organisms co-cultured with lymphocytes at 1:1 to 10:1 ratios generally induce optimal lymphocyte responses. Dattwyler and colleagues (1) applied this method to patients who were designated as having Lyme disease but were seronegative for B. burgdorferi antibodies. This diagnosis was based on previous erythema migrans, flu symptoms, residence in an endemic area, and low B. burgdorferi antibody levels in the setting of late disease. The mean response to B. burgdorferi in these patients was similar to that seen in seropositive patients with Lyme disease and was significantly greater than that seen in the healthy control group (P < 0.05). However, overlap with controls was seen within both patient groups. The apparent dissociation between humoral and cellular immunity was thought to be due to inadequate early anti288
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biotic treatment. Presumably, a reduction in antigen levels aborted maturation of a humoral response but not before the infection disseminated and T cells became sensitized. Our results confirm that, as a group, patients with Lyme disease have higher mean lymphocyte responses to B. burgdorferi than do healthy controls. Unfortunately, healthy controls also showed lymphocyte responses to B. burgdorferi and, in most cases, lymphocyte proliferation testing could not discriminate between the patient and the healthy control tested that same day. Lymphocyte stimulation by B. burgdorferi appears to have both specific and nonspecific features. The response to B. burgdorferi seen in immunologically naive fetal cord-blood lymphocytes suggests a nonspecific mitogen-like response. Sigal and colleagues (11) recently noted similar nonspecificity; in their study, lymphocytes from healthy controls produced increased IgM in vitro after B. burgdorferi stimulation. Increased antinuclear antibodies and rheumatoid factor rates reported in patients with Lyme disease may reflect polyclonal B-cell activation (12). A lipopolysaccharide isolated from B. burgdorferi has been reported to stimulate normal lymphocyte proliferation (13). Normal human monocytes release considerable interleukin-1 when exposed to B. burgdorferi, and lipopoly saccharide depletion by polymyxin does not affect this response (14). Once present, interleukin-1 could considerably augment other minor mitogenic stimulants. Our observation that the overall mean lymphocyte response seen in patients was significantly higher than the mean response seen in controls may indicate some degree of immune specificity. In addition, the individual responses seen in controls never exceeded those seen in patients. Efforts to improve this test's specificity by fractionating B. burgdorferi antigens are in progress (15, 16). The assay could also possibly be improved by simultaneously testing lymphocytes from a large panel of controls and patients known to have Lyme disease. Lymphocytes from patients might be preserved for culturing using cryopreservation technology. We hoped that lymphocyte testing might prove to be useful in ruling out exposure to B. burgdorferi. Based on our experience, lymphocyte proliferation induced by B. burgdorferi is possible regardless of exposure status, and a positive response by itself has little clinical significance. Clearly, interactions between B. burgdorferi and the immune system occur. The resultant immune response may be crucial in either resolving the infection or initiating pathologic changes with prolonged clinical manifestations (17-19). Until such studies are completed, however, we believe that lymphocyte response testing has little to add in either the diagnosis or the management of Lyme disease. Acknowledgments: The authors thank Dr. Russell Johnson, Department of Microbiology, University of Minnesota, for doing the ELISAs for B. burgdorferi and for providing the B. burgdorferi isolates. They also thank Leslie Deem and Joanne Bailey for secretarial assistance. Grant Support: In part by National Institutes of Health grant 5R01-AR33492. Requests for Reprints: David C. Zoschke, MD, Department of Medicine, Section of Rheumatology, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455.
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Current Author Addresses: Drs. Zoschke and Skemp and Mr. Defosse: Department of Medicine, Section of Rheumatology, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455. References 1. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golightly MG. Seronegative lyme disease: dissociation of the specific T- and B-lymphocyte responses to Borrelia burgdorferi. N Engl J Med. 1988;319:1441-6. 2. Steere AC. Lyme Disease. N Engl J Med. 1989;321:586-96. 3. Zoschke DC, Kaja J. Suboptimal levels of hydrogen peroxide scavengers in synovial fluid: in vitro augmentation with slow acting anti-rheumatic drugs. J Rheum. 1989;16:1233-40. 4. Steere AC, Grodzicki AN, Kornblatt AN, et al. The spirochetal etiology of Lyme disease. N Engl J Med. 1983;308:733-40. 5. Magnarelli LA, Anderson JF. Enzyme-linked immunosorbent assays for the detection of class-specific immunoglobulins to Borrelia burgdorferi. Am J Epidemiol. 1988;127:818-25. 6. Sigal LH. Lyme disease, 1988: immunologic manifestations and possible immunopathogenetic mechanisms. Semin Arthritis Rheum. 1989;18:151-67. 7. Schwartz BS, Goldstein MD, Ribeiro JMC, Schulze TL, Shahied SI. Antibody testing in Lyme disease. A comparison of results in four laboratories. JAMA. 1989;262:3431-4. 8. Benach JL, Coleman JL, Garcia-Monco JC, Deponte PC. Biological activity of Bb antigens. Ann NY Acad of Sci. 1988;539:115-25. 9. Schaible UE, Kramer MD, Justus CW, Museteneau C, Simon MM. Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi. Infect Immun. 1989;57:41-47.
10. Sigal LH, Steere AC, Freeman DH, et al. Proliferative response of mononuclear cells in Lyme disease. Arthritis Rheum. 1986;29:761-9. 11. Sigal LH, Steere AC, Dwyer JM. In vivo and in vitro evidence of B cell hyperactivity during Lyme disease. J Rheumatol. 1988;15:64854. 12. Goebel KM, Krause A, Neurath F. Acquired transient autoimmune reactions in Lyme arthritis: correlation between rheumatoid factor and disease activity. Scand J Rheum. 1988;75(Supp):314-7. 13. Beck G, Habicht GS, Benach JL, Coleman JL. Chemical and biologic characterization of a lipopolysaccharide extracted from the Lyme disease spirochete (Borrelia burgdorferi). J Infect Dis. 1985; 152:108-17. 14. Habicht GS, Beck G, Benach JL, Coleman JL, Leichtling KD. Lyme disease spirochetes induce human and murine interleukin-1 production. J Immunol. 1985;134:3147-54. 15. Yoshinari NH, Reinhardt BN, Steere AC. Components of B. burgdorferi causing T-cell proliferative responses in patients with Lyme arthritis [Abstract]. Arthritis Rheum. 1989;546:31. 16. Dattwyler R, Volkman P, Shepard D, Gorevic P. Characterization of the cell immune response to individual Borrelia burgdorferi antigens in Lyme borreliosis [Abstract]. Arthritis Rheum. 1989;80(Suppl):52. 17. Martin R, Ortlauf J, Sticht-Groh V, Bogjahn V, Goldman SF, Mertens HG. Borrelia burgdorferi-specific and autoreactive T-cell lines from cerebrospinal fluid in Lyme radiculomyelitis. Ann Neurol. 1988;24:509-16. 18. Sigal LH, Tatum AH. Tissue-specific auto-antibodies in a Lyme disease neurologic patient's sera bind to cross reacting B. burgdorferi and neuronal antigens. Arthritis Rheum. 1987;30:536. 19. Weyand LM, Goronzy JJ. Immune responses to Borrelia burgdorferi in patients with reactive arthritis. Arthritis Rheum. 1980;32:1057-64.
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