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CD4+ CD25+ T cells are a population of regulatory T cells associated with control of arthritis in anti-interleukin-17 antibody-treated Borrelia-vaccinated and challenged gamma interferon-deficient mice. Here, we present direct evidence that adoptive transfer of enriched CD4+ CD25+ T cells from these mice can prevent the development of arthritis in Borrelia-vaccinated and challenged mice. These findings establish a major role for CD4+ CD25+ T cells in the prevention of arthritis in Borrelia-vaccinated and challenged animals.
Recently, we showed that Borrelia-vaccinated and challenged gamma interferon-deficient (IFN-γ°) mice developed severe destructive osteoarthropathy (5). Development of arthritis in these mice was prevented by treatment with anti-interleukin-17 (anti-IL-17) antibody (3). Furthermore, anti-IL-17 treatment increased the number of CD4+ CD25+ T cells found in the regional lymph nodes of Borrelia-vaccinated and challenged IFN-γ° mice (16). When Borrelia-vaccinated and challenged mice were administered concomitantly anti-IL-17 and anti-CD25 antibodies, the number of CD4+ CD25+ T cells decreased in the lymph nodes (16). More importantly, severe destructive arthritis developed in the dual-antibody-treated Borrelia-vaccinated and challenged mice (16). These results suggest that CD4+ CD25+ T cells play a role in the prevention of arthritis associated with Borrelia-vaccinated and challenged IFN-γ° mice.
CD4+ CD25+ T cells are a naturally occurring, regulatory population of T cells responsible for down-regulation of immune responses for both foreign and self-antigens (1, 12, 19). These T cells have been shown to suppress or prevent graft-versus-host disease after allogeneic transplantation (10, 28, 32), abrogate the effects of insulin-dependent diabetes mellitus (6), block development of thyroiditis and colitis (11, 15), and control the severity of viral inflammatory lesions (23). Moreover, the significance of T regulatory CD4+ CD25+ cells in disease prevention and therapy has been demonstrated by transfer of cells into several animal disease models (18, 25, 31, 33). However, a definitive role for CD4+ CD25+ T cells in the regulation of arthritis associated with Borrelia-vaccinated and challenged mice has not yet been established. In this report, we provide evidence that CD4+ CD25+ T cells play a significant role in the prevention of arthritis associated with Borrelia-vaccinated and challenged IFN-γ° mice.
IFN-γ° gene-deficient mice (parental strain C57BL/6) were obtained from W. P. Weidanz (University of Wisconsin) with permission from Genentech, Inc. (South San Francisco, CA). The mice were bred at the animal facility located at the University of Wisconsin. Low-passage-number (<10) virulent Borrelia burgdorferi strain 297 (human spinal cord) and Borrelia bissettii (strain C-1-11, Microtus pennsylvanicus) were grown at 32°C in modified Barbour-Stoenner-Kelly (BSK) medium until reaching a concentration of approximately 107 spirochetes/ml. Methods for preparation of organisms and the whole-cell vaccine have been previously described (3, 5, 16). Whole cells of B. burgdorferi are not recommended for vaccination of humans because of previous concerns with other whole-cell vaccines (8). However, the ability of whole cells of B. burgdorferi to induce arthritis consistently in mice allows evaluation of immunological mechanisms responsible for the arthritis (3, 5, 16).
Twenty-one days after vaccination with B. burgdorferi isolate 297 in alum, mice were anesthetized with ether contained in a nose-and-mouth cup and injected subcutaneously in the left hind paws with 50 μl of BSK medium containing 106 viable B. bissettii (formerly B. burgdorferi isolate C-1-11) organisms. Swelling of the hind paws can also be induced with the homologous B. burgdorferi isolate 297. However, vaccinated mice must be challenged before protective antibodies develop (day 7) or after they have declined. Swelling of the hind paws of homologous vaccinated and challenged mice is varied. Therefore, we challenged B. burgdorferi isolate 297-vaccinated mice with B. bissettii to obtain consistent swelling of the hind paws.
Lyophilized rat anti-mouse IL-17 antibody (100 μg) was obtained from R&D Systems (Minneapolis, MN). The antibody was suspended in filter-sterilized (0.2-μm-pore-size filter; Acrodisk; Gelman Sciences, Ann Arbor, MI) phosphate-buffered saline (pH 7.4) to yield a concentration of 50 μg/ml. Twenty-one days after vaccination, mice were infected with B. bissettii in the hind paws. One hour after infection, the mice were injected in the hind paws with 50 μl of anti-IL-17 antibody. Antibody was injected daily thereafter for 7 days. Swelling of the hind paws of mice was used to evaluate the inflammatory response and has been previously described (3, 5, 16). The hind paw swelling among groups of mice was tested by analysis of variance. The alpha level was set at 0.05 before the experiments were started.
Eight days after Borrelia-vaccinated and challenged mice began treatment with anti-IL-17 antibody, the inguinal and popliteal lymph nodes were removed from the mice. Single-cell suspensions of the pooled lymph nodes were prepared by teasing apart the nodes with forceps and passing them through a sterile nylon mesh screen (Fisher, Hanover Park, IL) into cold Dulbecco's modified Eagle's medium (DMEM; Sigma). The inguinal and popliteal lymph node cells were dispensed into a 15-ml conical centrifuge tube (Fisher Scientific, Pittsburgh, PA) and mixed with 25 μl each of fluorescein isothiocyanate-conjugated rat anti-mouse CD4 antibody and R-phycoerythrin-conjugated rat anti-mouse CD25 antibody (BD PharMingen, San Diego, CA). The cells and antibodies were incubated at 4°C for 30 min under dark conditions. Isotype controls were also included.
Subsequently, the cells were washed by centrifugation with DMEM (4°C, 1,000 × g, 5 min) and the pellets were resuspended in 4 ml of cold DMEM. The cells were then separated into CD4+ CD25+ and CD4+ CD25− T-cell populations using a BD triple-laser FACSVantage SE (Becton Dickinson) and checked for purity using a FACSCalibur flow cytometer (Becton Dickinson) with CellQuest software (Becton Dickinson). The purities of the CD4+ CD25+ and CD4+ CD25− T-cell populations were 93% and 99%, respectively.
Recipient mice vaccinated with B. burgdorferi isolate 297 were infected with 106 B. bissettii organisms. Approximately 1 hour later, the Borrelia-vaccinated and challenged mice were injected with 0.5 unit of cobra venom factor anti-complementary protein (250 μg or 55 units; Sigma). The cobra venom factor anti-complementary protein was added to DMEM to yield a concentration of 10 units/ml. Fifty microliters (0.5 unit) was then injected into recipient mice. Cobra venom factor anti-complementary protein has been shown to cleave C3, leading to its depletion (29). Recipients were treated with cobra venom to minimize the lysis of CD4+ and CD25+ T cells that maintained antibody after flow cytometric processing. At 8 and 20 days after infection and cell transfer, mice were euthanized with ether and their hind paws were amputated at midfemur for histopathologic examination (3, 5, 16).
Inguinal and popliteal lymph nodes were obtained from eight anti-IL-17 antibody-treated Borrelia-vaccinated mice on day 8 after spirochetal infection (Fig. (Fig.1).1). We showed previously that the number of CD4+ CD25+ T cells (range, 3 × 105 to 7 × 105 cells/node) peaked on day 8 of infection or 24 h after completion of anti-IL-17 therapy (16). The nodes were pooled, and a suspension of single cells was obtained before they were processed by flow cytometry. Figure Figure1A1A shows the flow cytometric results after incubation of lymph node cells with fluorescein isothiocyanate-conjugated rat anti-mouse CD4 antibody and R-phycoerythrin-conjugated rat anti-mouse CD25 antibody. Approximately 4 × 105 CD4+ CD25+ T cells and 3 × 106 CD4+ CD25− T cells were detected per node. Upon completion of cell sorting enrichment (Fig. 1B and C), the purities of the CD4+ CD25+ T cells and CD4+ CD25− T cells were 93% and 99%, respectively.
Recipient Borrelia-vaccinated and challenged mice (four per group) treated with cobra venom anti-complementary protein were injected subcutaneously in the left hind paw with 1 × 105 immune CD4+ CD25+ T cells or CD4+ CD25− T cells and nonseparated immune lymph node cells. A fourth group of four Borrelia-vaccinated and challenged mice received no cells.
We showed previously that Borrelia-vaccinated and infected mice develop inflammation and edematous changes throughout the paws (3, 5, 16). This response began on days 4 to 6 after infection and peaked on day 8. In this study, Borrelia-vaccinated and infected mice that received no cells developed a similar response (Fig. (Fig.2).2). By contrast, Borrelia-vaccinated and infected mice infused with immune CD4+ CD25+ T cells, immune CD4+ CD25− T cells, or nonseparated immune lymph node cells developed more edema (P ≤ 0.05), specifically on days 6 through 10 after cell transfer. When these studies were repeated twice with four animals per group, similar responses occurred.
While only marginal differences in levels of swelling of the hind paws occurred among the recipients of CD4+ CD25+ T cells, CD4+ CD25− T cells, or nonseparated immune lymph node cells, significant differences in histopathologic changes in the ankle joints among these groups did occur 8 days after infection. Figure Figure33 shows the ankle joints of three different Borrelia-vaccinated and challenged mice treated with cobra venom and infused with CD4+ CD25+ T cells. The ankle joints of these CD4+ CD25+ T-cell recipients were free of histopathologic changes. Conversely, Fig. Fig.44 shows the histopathologic findings of Borrelia-vaccinated and challenged mice administered no cells (Fig. (Fig.4,4, top) or administered CD4+ CD25− T cells (Fig. (Fig.4,4, middle) or nonseparated immune cells (Fig. (Fig.4,4, bottom). Each of these panels shows inflammation of the subsynovial tissues surrounding the ankle joint, some destruction of articular cartilage, synovial hyperplasia, and infiltration of inflammatory cells, predominantly neutrophils, into the synovial space. Other recipients within these groups, although not uniform, developed destruction of the synovial lining or had occluded ankle spaces. When these studies were repeated twice, including with adoptive transfer of normal lymph node cells, similar results were obtained.
Twenty days after infection, no histopathologic changes were observed in the ankle joints of Borrelia-vaccinated and challenged mice treated with cobra venom and infused with CD4+ CD25+ T cells (Fig. (Fig.5,5, top left). By contrast, severe histopathologic changes were observed in recipients of CD4+ CD25− T cells (Fig. (Fig.5,5, top right) and nonseparated immune cells (Fig. (Fig.5,5, lower left) and in Borrelia-vaccinated and challenged mice receiving no cells (Fig. (Fig.4,4, lower right). Destruction of bone and cartilage occurred, and massive infiltration of inflammatory cells into the synovial space and subsynovial tissue was present. In other studies, normal cells were transferred, along with immune CD4+ CD25+ T cells, immune CD4+ CD25− T cells, or nonseparated immune cells. Similar results were detected.
Regulator cells like CD4+ CD25+ T cells have been demonstrated to suppress antigen-specific T-cell responses against allografts (32) and tumors (22) as well as bacterial (17), fungal (14), and parasitic (7, 13) antigens or infections. In addition, T regulatory cells have been implicated in the inability to clear infections (2), the prevention of vaccination responses (24), the control of autoimmune diseases (22), and the inhibition of fatal lymphoproliferative disease (9). CD4+ CD25+ T cells have also been implicated in the prevention of arthritis associated with Borrelia vaccination and infection (16).
In this report, we provide direct evidence that CD4+ CD25+ T cells can control the development of arthritis associated with Borrelia vaccination and challenge. Highly purified CD4+ CD25+ T cells (93%) were obtained from Borrelia-vaccinated and infected mice treated with anti-IL-17 antibody. Borrelia-vaccinated mice were infused with CD4+ CD25+ T cells following challenge. The vaccinated and challenged recipients failed to develop inflammation of the ankle joints, even 20 days after infection. In contrast, Borrelia-vaccinated and challenged recipients of CD4+ CD25− T cells developed considerable inflammation of the ankle joints. Similar findings were also observed when recipient Borrelia-vaccinated and challenged mice were injected with nonseparated immune lymph node cells, normal lymph node cells, or no cells. These results present compelling evidence that CD4+ CD25+ T cells can prevent the development of arthritis associated with an experimental model of Borrelia vaccination and infection.
The mechanism by which CD4+ CD25+ T cells accomplish this task is unknown. We hypothesize that CD4+ CD25+ T cells obtained from anti-IL-17-treated Borrelia-vaccinated and challenged mice prevents memory CD4+ T cells from responding to the spirochetal infection of Borrelia-vaccinated mice. A defining feature of CD4+ CD25+ T cells is their ability to prevent the activation and proliferation of CD4+ T cells (12, 21). Therefore, memory CD4+ T cells in recipients would not activate to cause the release of cytokines or other immune factors that culminates in the induction of arthritis. It is also possible that CD4+ CD25+ T cells exert deactivating or inhibiting effects (21) or down-regulate costimulatory molecules on antigen-presenting cells (4, 27). Any of these mechanisms would prevent the development of arthritis.
Although our histopathologic findings present compelling evidence that CD4+ CD25+ T cells are involved in the prevention of arthritis, measurement of hind paw swelling failed to separate recipients of CD4+ CD25+ T cells from control groups. Why did recipients of CD4+ CD25+ T cells develop edematous changes of the hind paws similarly to recipients of control cells? It is possible that different immune mechanisms are responsible for the edematous changes of the hind paws and the induction of arthritis. Weis et al. (30) also showed that edematous changes of the paws did not correlate with arthritis. Therefore, measurement of hind paw swelling cannot be used reliably to predict arthritis in mice.
Another question concerns whether CD4+ CD25+ T cells derived from vaccinated and infected mice without treatment with anti-IL-17 antibody or CD4+ CD25+ T cells derived from Borrelia-infected or normal mice can prevent arthritis. A preliminary study using normal CD4+ CD25+ T cells failed to prevent arthritis in adoptively immunized Borrelia-vaccinated and infected mice (data not shown). Several studies (20, 26) have reported that suppressor cells derived from normal mice are less efficient in preventing the development of autoimmunity. Antigen recognition may be required of some suppressor cells to exert their effect. We hypothesize that anti-IL-17 therapy increases pathogen-specific regulatory cells in Borrelia-vaccinated and challenged mice. Since anti-IL-17 therapy induces an inordinate number of CD4+ CD25+ T cells, it may also induce populations of novel regulatory cells that vary in function and are more efficient than normal or other immune CD4+ CD25+ T cells in preventing arthritis.
In conclusion, we showed that CD4+ CD25+ T cells can prevent arthritis induced in Borrelia-vaccinated and challenged animals in an experimental model. Additional studies are needed to define the mechanism by which CD4+ CD25+ T cells can prevent arthritis. This information may help to develop therapeutic agents to resolve Lyme arthritis in humans.
This study was supported by the Wisconsin State Laboratory of Hygiene; the public health laboratory for the state of Wisconsin, Madison, WI; and the Gundersen Lutheran Medical Foundation, La Crosse, WI.
We also thank the Flow Cytometry Facility at the University of Wisconsin Hospital (Madison, WI) for their assistance.