Factor Alpha by Borrelia burgdorferi

Transcription

1 INFECTION AND IMMUNITY, Mar. 1992, p /92/ $02.00/0 Copyright 1992, American Society for Microbiology Vol. 60, No. 3 In Vitro and In Vivo Induction of Tumor Necrosis Factor Alpha by Borrelia burgdorferi DANA L. DEFOSSE* AND RUSSELL C. JOHNSON Department of Microbiology, University of Minnesota, 420 Delaware Street, S.E., Box 196, Minneapolis, Minnesota Received 12 August 1991/Accepted 31 December 1991 Tumor necrosis factor alpha (TNF-at) is an immunoregulatory cytokine with many biological activities including the mediation of inflammation. We examined sera and synovial fluids from patients seropositive for infection with Borrelia burgdorfieri using a radioimmunoassay specific for TNF-a. Significant elevation of TNF-ce was found in the sera and synovial fluids of patients examined, while controls showed no elevation. Sera of mice infected with B. burgdorferi contained elevated levels of TNF-a which varied during the course of a 24-day infection. To determine whether B. burgdorferi is capable of inducing TNF-a production, spirochetes were added to adherent human peripheral blood mononuclear cells or mouse peritoneal exudate cells and 24 h later supernatants were assayed. TNF-oa induction occurred in a dose-dependent manner. The maximum stimulation occurred when a ratio of 1 to 10 spirochetes per mononuclear cell was used. At optimal concentrations, induction was not diminished by inactivation of spirochetes or pretreatment with polymyxin B. These results suggest that an increase in TNF-at production may occur as a result of infection with B. burgdorfieri. Lyme borreliosis is caused by the spirochete Borrelia burgdorferi (7) and is transmitted to humans primarily by ticks of the Ixodes ncinus complex (12). It is the most commonly reported arthropod-borne disease in the United States, where Lrodes dammini is the primary vector (39). This multisystem disorder occurs worldwide and is recognized as a disease of major significance. The first stage of Lyme borreliosis is localized and often begins as a rapidly expanding rash (erythema migrans) (2). Within weeks or months of onset, a second, disseminated stage which is characterized by neurological, musculoskeletal, and cardiac abnormalities may occur. These manifestations may collectively be referred to as early Lyme borreliosis (40). The third phase, or late Lyme borreliosis, may ensue months to years after infection. It is characterized by arthritis of the large joints (Lyme arthritis) or neurological abnormalities, either of which may be persistent (40). Tumor necrosis factor alpha (TNF-ct) is an immunoregulatory cytokine produced by mononuclear phagocytes and other cells (10). It was originally identified in the sera of mice sensitized with Mycobacterium bovis bacillus Calmette- Guerin (BCG) and challenged with lipopolysaccharide (LPS) (13) and was subsequently found to cause necrosis of murine and human tumors in mice (21). TNF-a has been implicated as the primary mediator in the pathogenesis of endotoxic shock (9, 29, 44). Elevated TNF-a levels have been demonstrated in the sera of patients with neoplasms (34), parasitic infections (14, 30, 34), AIDS (1, 25), and bacterial infections (45) including syphilis (34). Significant levels have also been demonstrated in the sera and synovial fluids of patients with rheumatoid, suppurative, or reactive arthritis (22, 31, 33). Recently, TNF-a has emerged as a pleiotropic biological mediator with many properties (for a review, see reference 37). Some bioactivities of this hormone may be relevant to inflammation and other events in the pathogenesis of diseases of connective tissue, including induction of interleukin * Corresponding author (IL-1) (28) and prostaglandin E2 (3), which results in the initiation of proteolysis. TNF-ot also has been shown to inhibit the biosynthesis of proteoglycan by chondrocytes and induce collagenase production by human synovial cells (15, 32). Other than IL-1, TNF-a is the only cytokine presently known to activate osteoclastic bone and cartilage resorption (8, 32, 43). To determine whether TNF-a is produced in response to B. burgdorfen, we examined the sera and synovial fluids of patients who were seropositive for infection with B. burgdorferi and the sera of infected mice. Significant levels of TNF-a were found in these specimens. Mononuclear cells from normal human donors and mice were found to produce TNF-co in a dose-dependent manner when cultured with spirochetes. MATERIALS AND METHODS Cells. Strain 297 of B. burgdorferi was isolated from human spinal fluid (39). Virulence was maintained by passage in hamsters, and the isolate was not subcultured in medium more than three times before use in the in vitro or in vivo assays. Spirochetes were grown in Barbour-Kelly- Stoenner (BSK) medium (4) at 30 C. Peritoneal exudate cells (PEC) and human peripheral blood mononuclear cells (PBMC) were cultured in RPMI 1640 (GIBCO Scientific, Grand Island, N.Y.) containing 10% fetal calf serum at 37 C in an atmosphere of 5% carbon dioxide. In some cases, penicillin (100 U/ml) and streptomycin (100,ug/ml) (GIBCO) were added to the medium. All glassware used were treated for endotoxin by dry baking at 450 C for 4 h. Fetal calf serum containing less than 1 ng of endotoxin per ml was obtained from GIBCO Scientific. Patient samples. Synovial fluids and sera were from Lyme borreliosis patients, patients with other forms of joint disease or arthritis, and healthy controls. Antibodies reactive with B. burgdorfen were determined with the enzyme-linked immunosorbent assay (ELISA) and by immunoblotting (26). Synovial fluids were aspirated from the knees of patients for

2 1110 DEFOSSE AND JOHNSON other diagnostic procedures or as part of normal therapy. All control synovial fluids and sera were shown to be seronegative by ELISA. All experiments using patient samples were approved by the University of Minnesota Committee on the Use of Human Subjects. TNF-ot measurement. TNF-ao levels in sera, synovial fluids, and culture supernatants were determined by radioimmunoassay (RIA) for human TNF-a or ELISA for murine TNF-a. Commercial kits were employed (Genzyme, Boston, Mass.), and the assay was performed according to the instructions provided. The RIA was liquid phase, using a rabbit polyclonal antibody specific for TNF-ao. The ELISA was a solid-phase assay utilizing a specific hamster monoclonal antibody for capture and polyclonal goat anti-tnf and horseradish peroxidase-conjugated donkey anti-goat antibodies for detection. All determinations were performed in duplicate. In vitro TNF-a production. PBMC were obtained from healthy donors (American Red Cross, St. Paul, Minn.) by separation on Ficoll-Hypaque gradients (11; Pharmacia Fine Chemicals, Piscataway, N.J.). The mononuclear fraction was obtained by adherence to plastic petri plates. Adherent cells were harvested with trypsin and characterized by myeloperoxidase staining (23) or by staining with a monoclonal antibody specific for marker M01 (Becton Dickinson, Mountain View, Calif.). A hemacytometer was used to calculate cell numbers, and viability was determined on the basis of trypan blue exclusion. Cells were plated in 24-well tissue culture plates in RPMI 1640 with 10% fetal calf serum, with or without antibiotics, at a concentration of 106 per well. After overnight incubation at 37 C, supernatants were removed and replaced with fresh medium alone, 1 jig of LPS (Sigma, St. Louis, Mo.) per ml or spirochetes, and incubated for an additional 24 h. Supernatants were then removed, centrifuged (10,000 x g for 10 min) and frozen at -20 C until assayed. Spirochetes were harvested by centrifugation of late-log-stage cultures (10,000 x g for 30 min), resuspended in RPMI 1640, and enumerated by using a Petroff-Hausser counting chamber and dark-field microscopy. Inactivated spirochetes were prepared by incubating overnight at 34WC in a 1:10,000 dilution of thimerosal and washing three times in RPMI Inactivation of spirochetes was confirmed by the absence of growth in BSK medium. Polymyxin B (PMB)- treated spirochetes were prepared by incubation for 1 h in RPMI 1640 containing 10,g of PMB per ml at 37 C. PEC were induced in BALB/c mice by injecting 1 ml of sterile thioglycolate broth (Difco, Detroit, Mich.). Two days later, PEC were collected by injecting 3 ml of RPMI 1640 intraperitoneally and aspirating cells. PEC were washed and processed as described above for PBMC. In vivo TNF-a production. BALB/c mice were injected intraperitoneally with ca. 108 B. burgdorfen 297. Subsequently, two mice were selected at random and serum samples were collected by cardiac puncture. Blood samples and a 1:10 homogenate of tissues from mice were cultured in BSK medium to confirm infection with B. burgdorferi. RESULTS TNF-a levels in patient sera and synovial fluids. To determine whether elevation of TNF-ao occurs during Lyme borreliosis, sera and synovial fluid from patients seropositive for B. burgdorfen were examined by RIA (Fig. 1). Of 30 patient serum samples, 8 (26.6%) were below the detection limit of the assay (150 pg/ml). TNF-a was elevated in 22 (73.3%) patient serum samples. Moderate elevation (200 to pg/ul TNFa * OA * AA INFECTr. IMMUN. CONTROL PATIENT CONTROL PATIENT SERA SERA SYN FLUID SYN FLUID LIMIT FIG. 1. Samples of serum from patients seropositive for infection with B. burgdorferi and synovial (syn) fluid from patients with Lyme arthritis or other nonrheumatoid arthritis were examined for TNF-at using a RIA. Control synovial fluids used were as follows: avascular necrosis (A), gout (G), nonrheumatoid arthritis of unknown etiology (N), osteoarthritis (0), and chronic synovitis (C). 500 pg/ml) was seen in 10 samples, and marked elevation (>500 pg/ml) was detected in 12 samples. The range was 100 to 1,250 pg/ml, and the median value was 382 pg/ml. Control sera from healthy, seronegative donors (n = 6) contained no detectable TNF-ao. No correlation was seen between TNF-a levels and antibody levels to B. burgdorfeti (data not shown). TNF-ao levels of patient synovial fluids (n = 9) ranged from 150 to 1,280 pg/ml. While three samples were within the range of the controls, all six elevated samples contained greater than 500 pg/ml. Control synovial fluids (n = 7) were selected from patients with noninfectious, nonrheumatoid arthritis. The following values of TNF-a (in picograms per milliliter) were found in the seven synovial fluid controls examined; 600 in one patient diagnosed with gout, 360 in one patient with chronic synovitis, 560 in one patient with a diagnosis of avascular necrosis, and <150 from three osteoarthritis patients and one patient with nonrheumatoid arthritis of unknown etiology. The absence of antibody to B. burgdorfei was confirmed by ELISA in all control synovial fluids. In vitro stimulation of PBMC and PEC. Stimulation of the PBMC of healthy human donors by B. burgdorferi 297 for 24 h elicited a dose-dependent cellular response, resulting in the production of TNF-(x levels higher than those in control cultures (Fig. 2). For the three representative donors shown, maximum stimulation occurred when a ratio of 1 to 10 spirochetes per PBMC was used. The presence of the spirochetes was not toxic for the PBMC or PEC, and at least 80% were shown to be viable by trypan blue exclusion after 24 h of coincubation while spirochete motility was reduced to 50% or greater. At optimal effector ratios, spirochetes induced TNF-cx levels in PBMC and PEC comparable to those achieved with LPS (Table 1). B. burgdorferi inactivated by thimerosal showed equivalent effectiveness in stimulating the release of TNF-a from human PBMC as that of living spirochetes. Pretreatment of spirochetes with 10,ug of PMB per ml did not diminish their ability to stimulate mononuclear cells. PMB alone did not induce TNF-a production by PBMC. In the absence of PBMC or PEC, spirochetes, LPS or PMB were not reactive in either TNF assay. Serum TNF-a during murine borreliosis. Mice with ongo-

3 VOL. 60, 1992 TNF-ot INDUCTION BY B. BURGDORFERI O Donor 3 C 1200 U3 Donor 2wT_ OL 800- a -Ec; -s c Is DISCUSSION The suggestion that TNF-ot is induced during infection with B. burgdorfen is supported by the results of in vivo and in vitro experiments. Lyme borreliosis patient serum and synovial fluids (Fig. 1) were shown to contain high levels of detectable TNF-a in some cases. These findings are comparable to those seen in patients with rheumatoid, suppurative, and reactive arthritis (22, 31, 33). Elevation was seen with both sera and synovial fluids of patients with Lyme borreliosis. The median level in synovial fluids (570 pg/ml) was greater than that in sera (382 pg/ml). Previous studies of arthritis patients differ in the ability to demonstrate TNF-a in both sera and synovial fluids. Saez- Llorens et al. (31) found that although suppurative arthritis patients had excessive quantities of TNF-a in synovial fluids, they were unable to detect this cytokine in sera. However, Saxne et al. (33) showed that TNF-a was highly elevated in simultaneously collected sera and synovial fluids from several patients with rheumatoid arthritis. Although the samples examined in this study were not matched, our ' ' T1 ony only 4 Concentration spirochetespog /ml) FIG. 2. Adherent PBMC from three healthy human donors were isolated by Ficoll gradient centrifugation and adherence to plastic. Cultures (1 ml) containing 106 PBMC were incubated with various concentrations of live virulent B. burgdorfen. After 24 h, cell-free supernatants were assayed for TNF-a by RIA. ing B. burgdorferi infection produced substantial levels of serum TNF-a with the highest levels observed during the sixth day of infection (Fig. 3). Spirochetes were isolated from blood samples from 4 to 6 days after infection and from the tissues throughout the 24-day duration of the experiment. o Day FIG. 3. BALB/c mice were infected with 10' B. burgdorferi 297. At the indicated intervals, two mice were selected at random and euthanized and blood samples were collected by cardiac puncture. Sera from mice were pooled, and TNF-a levels were determined by ELISA. Values represent means + standard deviations of duplicate determinations of the pooled sera. findings support the contention that patients with Lyme borreliosis may possess significant levels of detectable TNF-a in sera. Our study and that of Saxne et al. (33) used immunological assays for detection of TNF-a, while that of Saez-Llorens (31) employed a bioassay. A possible explanation for the differences observed in these results could be that TNF-ot released into the serum is inactivated by inhibitors (19, 35, 36) and therefore not detected in biological assays. The observation that significant levels of TNF-a can be detected in sera of patients infected with B. burgdorfen and is present in synovial fluids of Lyme arthritis patients supports a role for this cytokine in some facet of the pathogenesis of this disease. Relevant properties of this hormone include the induction of IL-1 secretion by mononuclear cells (28), stimulation of bone resorption (8, 44), induction of collagenase and prostaglandin secretion by synoviocytes (15), inhibition of synthesis and resorption of proteoglycans of cartilage (32), fibroblast proliferation (16, 41), and endothelial cell activation (17). Elevated prostaglandin E2 and collagenase levels have been demonstrated in Lyme arthritis patients (38). Prostaglandin E2 is thought to stimulate the release of intracellular proteases which can result in degradation of joint tissue and impede remodeling (15). Collagenase disrupts the extracellular matrix of joint tissues resulting in increased induration. Elevated levels of TABLE 1. TNF-a production by mononuclear cells Mean amt (pg/ml) of TNF-aL produced ± SE' Condition Human PBMC donor: BALB/c mice PEC Medium alone ± 28 LPS (1.0,ug/ml) 725 ± 218 1,425 ± 150 NDb Spirochetes alone 1,680 ± ± ,525 ± 154 Inactivated spirochetes 1, ,125 ± ± 318 ND PMB-treated spirochetesc 1,425 ± 725 1, , ND a Values for PBMC values are from representative donors and PEC values are from two separate experiments. Supernatants from duplicate or triplicate wells were assayed in duplicate. b ND, not determined. c Spirochetes were preincubated with 10 ilg of PMB per ml.

4 1112 DEFOSSE AND JOHNSON IL-1 have been demonstrated in synovial fluids from Lyme arthritis patients (5), and IL-1 is produced by macrophages in response to B. burgdorfeni in vitro (5, 20). Human PBMC and PEC produced TNF-ot in response to stimulation by B. burgdorferi and endotoxin. The response of human PBMC was shown to be dose dependent. Although responses varied among donors, a relatively small stimulus (1 to 100 spirochetes per PBMC) was capable of initiating a substantial production of TNF-ao comparable to that achieved with endotoxin, a potent stimulator of TNF-ao secretion. The ability of inactivated spirochetes to elicit responses equivalent to that of viable cells indicates that bacterial biosynthesis and motility are not required for induction of TNF-ot. Whether LPS is a cellular component of B. burgdorferi is presently the subject of debate (6, 42). Several lines of evidence suggest that an LPS-like material exists that possesses some activities of classical LPS (6, 18, 46). Our results show that PMB did not inhibit TNF-a induction by B. burgdorfen. This antibiotic has been shown to inhibit mitogenic activity of B. burgdorfen components (5) but not its ability to induce IL-1 (20). The biological activities of some forms of bacterial LPS are not inhibited by PMB (24, 27). Mice infected with B. burgdorfeni demonstrated substantial elevation of serum TNF-a. The isolation of spirochetes from blood samples of mice during the first 6 days supported the observed kinetics of serum TNF-at elevation. Demonstration of high concentrations of TNF-a in the sera of infected mice for 2 months concurs with the observation of sustained elevated levels in patients. The observation of substantially elevated levels of TNF-a in sera and synovial fluids of Lyme borreliosis patients and sera of experimentally infected mice provides evidence that this cytokine is produced during infection with B. burgdorferi. Similar levels of bioactive TNF-ot have been observed in other disease states including syphilis and arthritis (1, 20, 22, 25, 30, 31, 33, 34, 46). The potential role of TNF-ax in the pathogenesis of Lyme arthritis suggests that further investigation is warranted. This information, coupled with an emerging understanding of TNF-a regulation and pathophysiology, could contribute to diagnostic and therapeutic advances. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AR from the National Institute of Health to R.C.J. We thank Elizabeth Arendt for providing synovial fluids. Brenda Evangelista and Leann Husband provided technical assistance. REFERENCES 1. Ammann, A. J., M. A. Palladino, P. Volberding, D. Abrams, N. L. Martin, and M. Conant Tumor necrosis factor alpha and beta in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. J. Clin. Immunol. 7: Asbrink, D., and A. Homark Early and late cutaneous manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis). Ann. N.Y. Acad. Sci. 539: Bachwich, P. R., S. W. Chensue, J. W. Larrick, and S. L. Kunkel Tumor necrosis factor stimulates interleukin-1 and prostaglandin E2 production in resting macrophages. Biochem. Biophys. Res. Commun. 136: Barbour, A. G Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57: Beck, G., J. L. Benach, and G. S. Habicht Isolation of interleukin-1 from joint fluids of patients with Lyme disease. J. Rheumatol. 16: Beck, G., G. S. Habicht, J. L. Benach, and J. L. Coleman INFECT. IMMUN. Chemical and biological characterization of a lipopolysaccharide extracted from the lyme disease spirochete (Borrelia burgdorfen). J. Infect. Dis. 152: Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G. Barbour, W. Burgdorfer, R. Eldman, and R. A. Kaslow Spirochetes isolated from the blood of two patients with Lyme disease. N. Engl. J. Med. 308: Bertolini, D. R., G. E. Nedwin, T. S. Bringman, D. D. Smith, and G. R. Mundy Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factors. Nature (London) 319: Beutler, B., I. W. Milsark, and A. Cerami Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science 229: Beutler, B. J., J. Mahoney, J. LeTrang, P. Pekala, and A. Cerami Purification of cachectin, a lipoprotein lipase suppressing hormone secreted by endotoxin induced RAW cells. J. Exp. Med. 161: Boyum, A Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. Suppl. 20: Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis Lyme disease: a tick-borne spirochetosis? Science 216: Carswell, E. A., L. J. Old, R. L. Kassel, S. Green, N. Fiore, and B. Williamson An endotoxin-induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. USA 72: Clark, I. A., and G. Chaudhri Tumor necrosis factor in malaria induced abortion. Am. J. Trop. Med. Hyg. 39: Dayer, J. M., B. Beutler, and A. Cerami Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J. Exp. Med. 162: Fransen, L. J., J. Van der Heyden, R. Ruysschaert, and W. Fiers Recombinant tumor necrosis factor: its effects and its synergism with interferon--y on a variety of normal and transformed cell lines. Eur. J. Cancer Res. Clin. Oncol. 22: Frater-Schroeder, M., W. Risau, R. Hallmann, P. Gautschi, and P. Bohlen Tumor necrosis factor type a, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc. Natl. Acad. Sci. USA 84: Fumarola, D., I. Munno, and G. Miragliotta Endotoxicity of the Lyme disease spirochete. Infection 11: Gatanaga, T., R. Lentz, I. Masunaka, J. Tomich, E. W. Jeffes, M. Baird, and G. A. Granger Identification of TNF-LT blocking factor(s) in the serum and ultrafiltrates of human cancer patients. Lymphokine Res. 9: Habicht, G. S., G. Beck, J. L. Benach, J. L. Coleman, and K. D. Leichtling Lyme disease spirochetes induce human and murine IL-1 production. J. Immunol. 134: Haranaka, K. H., N. Satomi, and A. Sakrai Antitumor activity of murine tumors and heterotransplanted human tumors in nude mice. Int. J. Cancer 34: Hopkins, S. J., and A. Meager Cytokines in synovial fluid: the presence of tumor necrosis factor and interferon. Clin. Exp. Immunol. 73: Kaplow, L. S Simplified myeloperoxidase stain using benzidione dihydrochloride. Blood 26: Koga, T., T. Nishihara, T. Fujiwara, T. Nisizawa, N. Okahashi, T. Noguchi, and S. Hamada Biochemical and immunobiological properties of lipopolysaccharide (LPS) from Bacteroides gingivalis and comparison with LPS from Eschenichia coli. Infect. Immun. 47: Lahdevirta, J., C. P. J. Maury, A. M. Teppo, and H. Repo Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome. Am. J. Med. 85: Magnarelli, L. A., J. M. Meegan, J. F. Anderson, and W. A. Chappell Comparison of an indirect fluorescent-antibody test with an enzyme-linked immunosorbent assay for serological studies of Lyme disease. J. Clin. Microbiol. 20:

5 VOL. 60, 1992 TNF-a INDUCTION BY B. BURGDORFERI Mansheim, B. J., A. B. Onderdonk, and D. L. Kasper Immunochemical and biologic studies of the lipopolysaccharide of Bactroides melaninogenicus subspecies asaccharolyticus. J. Immunol. 120: Nawroth, P. P., I. Bank, D. Handley, J. Cassimeris, L. Chess, and D. Stern Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin-1. J. Exp. Med. 163: Opal, S. M., A. S. Cross, N. M. Kelly, J. C. Sadoff, M. W. Bodmer, J. E. Palardy, and G. H. Victor Efficacy of a monoclonal antibody directed against tumor necrosis factor in protecting neutropenic rats from lethal infection with Pseudomonas aeruginosa. J. Infect. Dis. 161: Pisa, P., M. Gennene, 0. Soder, T. Ottenhoff, M. Hansson, and R. Kiessling Serum tumor necrosis factor levels and disease dissemination in leprosy and leishmaniasis. J. Infect. Dis. 161: Saez-Llorens, X., N. M. Mustafa, 0. Ramilo, C. Fink, B. Beutler, and J. D. Nelson Tumor necrosis factor a and interleukin 1B in synovial fluid of infants and children with suppurative arthritis. Am. J. Dis. Child. 144: Saklatvala, J Tumor necrosis factor a stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature (London) 322: Saxne, T., M. A. Palladino, Jr., D. Heinegard, N. Talal, and F. A. Wollheim Detection of tumor necrosis factor a but not tumor necrosis factor P in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum. 31: Scuderi, P., K. S. Lam, K. J. Ryan, E. Petersen, K. E. Sterling, P. R. Finley, G. C. Ray, D. J. Slymen, and S. E. Salmon Raised serum levels of tumor necrosis factor in parasitic infections. Lancet ii: Seckinger, P., E. Vey, G. Turcatti, P. Wingfield, and J. M. Dayer Tumor necrosis factor inhibitor: purification, NH2- terminal amino acid sequence and evidence for anti-inflammatory and immunomodulatory activities. Eur. J. Immunol. 20: Seckinger, P., J. H. Zhang, B. Haupymann, and J. M. Dayer Characterization of a tumor necrosis factor alpha (TNFalpha) inhibitor: evidence of immunological cross reactivity with the TNF receptor. Proc. Natl. Acad. Sci. USA 87: Sherry, B., and A. Cerami Cachectin/tumor necrosis factor exerts endocrine, paracrine and autocrine control of inflammatory responses. J. Cell Biol. 107: Steere, A. C., C. E. Brinckerhoff, D. J. Miller, H. Drinker, E. D. Harris, and S. E. Malawista Elevated levels of collagenase and prostaglandin E2 from synovium associated with erosion of cartilage and bone in a patient with chronic Lyme arthritis. Arthritis Rheum. 31: Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308: Steere, A. C., S. E. Malawista, J. A. Hardin, S. Ruddy, P. W. Askenase, and W. A. Andiman Erythema chronicum migrans and Lyme arthritis-the enlarging clinical spectrum. Ann. Intern. Med. 86: Sugarman, B. J., B. B. Aggarwal, P. E. Hass, I. S. Figar, M. A. Paladino, Jr., and H. M. Shepard Recombinant human tumor necrosis factor-a: effects on proliferation of normal and transformed cells in vitro. Science 230: Takayama, K., R. J. Rothenberg, and A. G. Barbour Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 55: Thomson, B. M., G. R. Mundy, and T. J. Chambers Tumor necrosis factors alpha and beta induce osteoclastic bone resorption. J. Immunol. 138: Tracey, K. J., Y. Fong, D. G. Hesse, K. R. Manogue, A. T. Lee, G. C. Kuo, S. F. Lowry, and A. Cerami Anti-cachectin/ TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature (London) 330: Waage, A., A. Halstensen, and T. Espevik Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet i: Wright, D. J. M Reaction following treatment of murine borreliosis and shwartzman type reaction with borrelial sonicates. Parasite Immunol. 2: