Cytokines in autoimmune lacrimal gland disease in MRL/MpJ mice.

PURPOSE: MRL/MpJ-+/+ (MRL/+) and MRL/MpJ-lpr/lpr (MRL/lpr) mice show spontaneous development of a T-cell-driven lacrimal gland inflammation that is a model for Sjögren syndrome. The lacrimal gland lesions in these mice were evaluated by quantitative RT-PCR for selected cytokine mRNA for the relative contributions of T-helper (Th)1 versus Th2 immune responses and by RT-PCR and immunohistochemistry for the contribution of the interleukin (IL)-2/IL-2 receptor (IL-2R) autocrine pathway. METHODS: RNA was isolated from lacrimal glands of MRL/+ mice ages 1 to 9 months and from MRL/lpr mice ages 1 through 5 months, and competitive RT-PCR was used to quantify mRNA for the cytokines IL-2, -4, -10, and -12 and interferon (IFN)-gamma. Frozen sections of lacrimal glands from MRL/+ and MRL/lpr mice ages 2 through 5 months were stained for the IL-2R. RESULTS: IL-2 and -12 mRNA transcripts were below the limit of detection (<10(-3) fg/pg hypoxanthine phosphoribosyl transferase gene; HPRT) in both MRL/+ and MRL/lpr mice of all ages. When detectable, IFN-gamma transcripts were present in low amounts and were below the limit of detection in most samples. IL-4 transcripts were present in 100- to 1000-fold greater amounts than IFN-gamma transcripts. IL-10 transcripts were detectable in both MRL/+ and MRL/lpr mice. IL-2R typically was detected on less than 10% of lymphocytes infiltrating lacrimal gland lesions in both substrains. CONCLUSIONS: On the basis of RT-PCR for cytokine mRNA, autoimmune lacrimal gland lesions in MRL/+ and MRL/lpr mice appear to be largely Th2-mediated. There does not appear to be a direct role for the IL-2/IL-2R autocrine pathway within the microenvironment of the lacrimal gland.

The role of Fas-Fas ligand-mediated apoptosis in autoimmune lacrimal gland disease in MRL/MpJ mice.

PURPOSE: MRL/MpJ mice spontaneously develop lacrimal gland inflammation and are a model for the human disorder Sjögren's syndrome. MRL/MpJ-lpr/lpr (MRL/lpr) and MRL/Mp-+/+ (MRL/+) mice are congenic substrains, which differ only by a single autosomal recessive gene, the lpr mutation. This mutation results in defective Fas protein, defective lymphocytic apoptosis, and accelerated autoimmune lacrimal gland disease in MRL/lpr mice. We evaluated apoptosis in the lacrimal glands of MRL/lpr and MRL/+ mice. METHODS: Inflammatory cells in the lacrimal glands of MRL/lpr and MRL/+ mice were evaluated for apoptosis with TUNEL staining and Fas and Fas ligand expression with immunohistochemistry. RESULTS: MRL/lpr mice had a greater percentage of the lacrimal gland replaced by inflammatory infiltrate (30.3% +/- 7.0%) than did MRL/+ mice (13.0% +/- 3.0%, P = 0.02). However, similar amounts of lymphocytic apoptosis were present in the lacrimal glands of MRL/lpr and MRL/+ mice. The mean number of apoptotic cells per unit area of inflammation was 23.8 +/- 2.4 in MRL/lpr mice and 24.6 +/- 6.0 in MRL/+ mice (P = 0.91). Fas expression was absent on lymphocytes in MRL/lpr mice but was present on lymphocytes in MRL/+ mice. Fas ligand expression was present on epithelial structures in both substrains. CONCLUSIONS: The accelerated lacrimal gland disease inflammation in MRL/lpr mice does not appear to be due to decreased apoptosis in the microenvironment of the lacrimal gland of MRL/lpr mice. It appears that in MRL/lpr mice there is defective extrathymic lymphoid apoptosis, permitting a relatively greater expansion of autoreactive T cells, which subsequently invade the lacrimal gland.

Th1 versus Th2 immune responses in autoimmune lacrimal gland disease in MRL/Mp mice.

PURPOSE: In MRL/Mp-lpr/lpr (MRL/lpr) and MRL/Mp-+/+ (MRL/+) mice, a T-cell-driven lacrimal gland inflammation spontaneously develops that is a model for Sjögren's syndrome. The lacrimal gland lesions in these mice were evaluated by immunohistochemistry for the relative contributions of T-helper (Th)1 versus Th2 immune responses. METHODS: Frozen sections of lacrimal glands from MRL/lpr and MRL/+ mice ages 1 through 5 months were stained with monoclonal antibodies to the cytokines interferon (IFN)-gamma and interleukin (IL)4 and to the cell surface costimulatory molecules B7-1 and B7-2, which are associated with Th1 and Th2 responses, respectively. RESULTS: The median proportion of cells staining for IL-4 ranged from 30% to 67% over time for MRL/lpr mice and from 30% to 55% for MRL/+ mice. The median proportion of cells staining for IFN-gamma ranged from 1% to 5% for MRL/lpr mice and from 0% to 3% for MRL/+ mice. The proportion of cells staining positively for IL-4 was significantly greater than for IFN-gamma in both MRL/lpr (mean difference, 33%; P = 0.0001) and MRL/+ mice (mean difference, 42%; P = 0.0002). The median proportion of cells staining positively for B7-2 ranged from 20% to 38% for MRL/lpr mice and from 16% to 34% for MRL/+ mice. The median proportion of cells staining for B7-1 ranged from 2% to 10% for MRL/lpr mice and from 2% to 5% for MRL/+ mice. The proportion of cells staining positively for B7-2 was significantly greater than for B7-1 for both MRL/lpr mice (mean difference, 15%; P = 0.001) and for MRL/+ mice (mean difference, 19%; P = 0.006). CONCLUSIONS: On the basis of immunohistochemistry for cytokines and costimulatory molecules, inflammatory lacrimal gland lesions in MRL/lpr and MRL/+ mice appear to be a largely Th2 phenomenon.

T cell traffic and the inflammatory response in experimental autoimmune uveoretinitis.

PURPOSE: To quantify S-antigen-specific (S-Ag) T cells in the retina after adoptive transfer, and to evaluate their role in the initiation and progress of destructive ocular inflammation in experimental autoimmune uveoretinitis (EAU). METHODS: Lewis rats were administered 10 x 10(6) S-Ag-specific T cells from the SP35 cell line or 10 x 10(6) concanavalin A-stimulated syngeneic spleen cell lymphoblasts labeled with lipophilic PKH26 fluorescent dye immediately before intravenous inoculation. Labeled cells in each retina were counted at various times from 4 to 120 hours after cell transfer by fluorescence microscopic analysis of each dissociated retina. Recipient eyes were examined within the same period by light and confocal microscope. RESULTS: SP35 T cells showed a biphasic distribution in the retina. The first peak of 160 cells/retina was noted at 24 hours. A steady decline of labeled cells at 48 and 72 hours was followed by a rapid increase at 96 and 120 hours. Concanavalin A-stimulated, control-labeled cell populations showed an identical peak at 24 hours but a persistent decline thereafter; only two or three T cells were present in each retina at 120 hours. Concurrent inoculation of SP35 cells and nonspecific T cell blasts did not produce more SP35 cells than control cells in the retina at any time. Microscopic analysis showed mononuclear cell infiltration of the iris, ciliary body, and aqueous humor at 48 hours, which intensified rapidly and persisted through 120 hours. Retinal inflammation did not begin until 80 hours. Mononuclear cell adherence to vascular endothelium and perivascular macrophage infiltration of the innermost layers progressed to edema, and profound destructive inflammation and loss of retinal stratification were observed at 120 hours. CONCLUSIONS: There is no evidence of a blood-ocular or blood-retinal barrier to activated T cell blasts. Autologous S-Ag does not provoke a more rapid entry of specific T cells at that site. The data confirm that anterior segment inflammation precedes retinal inflammation, even though S-Ag-specific T cells were present in the retina within a few hours after cell transfer. Because S-Ag is clearly present in the retina, delay in antigen presentation at that site may account for the temporal difference between retinal and anterior segment inflammation.

Role of T cells in the pathogenesis of autoimmune lacrimal gland disease in MRL/Mp-lpr/lpr mice.

PURPOSE: MRL/Mp-lpr/lpr mice (MRL/lpr) spontaneously develop an autoimmune disease, including lacrimal gland lesions, which are a model for Sjögren's syndrome. Target organ lesions in MRL/lpr mice are composed largely of CD4+ T cells, and treatment with monoclonal antibodies (mAb) against CD4 improves in the systemic autoimmune disease but not the lacrimal gland inflammation. In anti-CD4 mAb-treated MRL/lpr mice, the lacrimal gland lesions are composed largely of CD8+ T cells. The effects of depletion of: (1) all T cells; (2) both CD4+ and CD8+ T cells, and (3) only CD8+ T cells on the lacrimal gland disease were investigated. METHODS: MRL/lpr mice underwent neonatal thymectomy and were treated with weekly injections of 6 mg of anti-Thy 1 mAb from age one week until sacrifice at age five months. Control nonthymectomized mice underwent similar treatment with either saline or normal rat immunoglobulin (rIg) injections. In a second experiment, MRL/lpr mice were treated with weekly injections of either: (1) 2 mg anti-CD4 mAb and 5 mg anti-CD8; or (2) 5 mg anti-CD8 alone. Control mice underwent similar treatment with either saline or rIg injections. RESULTS: Combined treatment with neonatal thymectomy and anti-Thy 1 mAb was effective in reducing the lacrimal gland disease in both frequency (50% > or = grade 3 vs. 100% in controls, P < 0.002) and extent (median 0% of lacrimal gland area involved by inflammation vs. 14.8% in controls; P = 0.01). Combined anti-CD4 and anti-CD8 therapy also was effective in reducing the lacrimal gland disease in terms of frequency (25% grade 3 vs. 93% in controls; P = 0.002) and extent (median 0% of lacrimal gland involved by inflammation vs. 12.9% in controls; P = 0.0005). Treatment with anti-CD8 mAb therapy alone was ineffective. The systemic autoimmune disease was also improved by T cell depletion and by combined anti-CD4 and anti-CD8 mAb therapy but not by anti-CD8 mAb therapy alone. CONCLUSIONS: Suppression of both CD4+ and CD8+ T cells is required to suppress lacrimal gland inflammation in MRL/lpr mice.

Biochemical and functional antigenic mimicry by a polyclonal anti-idiotypic antibody for chlamydial exoglycolipid antigen.

A vaccine developed by using a genus-specific antigen (Ag) of Chlamydia trachomatis would elicit a wide range of protection against various chlamydial infections. We have produced an anti-idiotypic antibody (Ab2) in guinea pigs which, in rabbits, mimics the immunogenicity of a genus-specific exoglycolipid Ag (GLXA) of C trachomatis. Furthermore, the Ab2 fulfills the functional criteria of an 'internal image' of the nominal Ag: it inhibits the binding of the idiotypic (Id) monoclonal Ab (mAb1) to GLXA, and it induces in rabbits anti-anti-Id antibody (Ab3) which recognizes both the affinity-purified nominal Ag GLXA and whole organisms. Moreover, Ab3 induced by immunization of rabbits with guinea pig Ab2 neutralizes infectious heterologous chlamydiae and prevents in vitro and in vivo infection. Taken together, these results demonstrate functional and biochemical mimicry of the Ab2 for the chlamydial GLXA and suggest that anti-idiotypic Ab to GLXA is a potential candidate vaccine against chlamydia-related diseases.

Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection.

Chlamydia trachomatis is the leading cause worldwide of preventable infectious blindness (trachoma) and sexually transmitted disease, including nongonoccocal urethritis and pelvic inflammatory disease. To date, no effective vaccine against C. trachomatis infection has been identified. A monoclonal anti-idiotypic antibody (anti-Id) to the chlamydial exoglycolipid antigen (GLXA) was tested in a murine model of ocular chlamydial infection for its ability to induce systemic immunity, which reduces microbiologic and clinical disease. The anti-Id to GLXA, delivered either systemically in soluble form or orally after encapsulation in poly(lactide) microspheres, induced significant protective immunity against ocular challenge of mice with a human biovar of C. trachomatis. Protection was associated with induction of anti-GLXA antibody and anti-chlamydial neutralizing antibody.

Intrastromal epithelial accretion follows deep excimer annular keratectomy.

The purpose of this study was to evaluate corneal reepithelialization and wound healing following annular excimer keratectomy. Two sets of experiments were performed on 35 rabbit eyes. In the first set of experiments, experiment I, deep Fresnel excimer keratectomy was performed, with a 6-mm outer and 3-mm inner diameter. Animals were sacrificed at 1, 5, 12, and 16 weeks, and corneas were examined by light and electron microscopy. In experiment II, the central epithelium was left intact, and superficial and deep mid-peripheral excimer annular keratectomies were performed measuring 6 mm in outer and 3 mm in inner diameter. Animals were sacrificed at 1 week, and corneas were examined by light microscopy. Following deep Fresnel excimer keratectomy (experiment I), corneas showed stromal edema in the central 3-mm zone. Intrastromal islands of epithelial cells with PAS positive basement membrane-like structures were seen histologically at 1 week. Electron microscopy showed loss of stromal collagen in areas adjacent to epithelial islands; in areas distant from the epithelial islands, the stromal collagen appeared normal. The overlying central stroma sloughed after 5 weeks. Anterior stromal scarring was observed. In experiment II (mid-peripheral annular keratectomy), intrastromal epithelial accretion was present in corneas with deep annular keratectomy but not in superficial annular keratectomy. Intrastromal epithelial accretion follows deep excimer annular keratectomy and is associated with adjacent stromal degradation.

Cyclosporine therapy suppresses ocular and lacrimal gland disease in MRL/Mp-lpr/lpr mice.

PURPOSE: MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop an autoimmune disease characterized by lymphoproliferation, vasculitis, glomerulonephritis, autoantibody production, and ocular and lacrimal gland inflammation. Lacrimal gland lesions in MRL/lpr mice are a model for the human disorder Sjögren's syndrome. The target organ lesions in MRL/lpr mice, including those in the eye and lacrimal gland, are composed largely of CD4+ T cells, with lesser numbers of CD8+ T cells and B cells. Cyclosporine therapy was evaluated for its effect on the autoimmune disease, particularly in the eye and lacrimal gland. METHODS: MRL/lpr mice were administered cyclosporine intraperitoneally at a dosage of 2 mg daily from age 1 to 5 months. Animals were killed at 5 months and evaluated for the presence of autoimmune disease. Control groups consisted of animals given daily injections with either saline or the cyclosporine diluent. RESULTS: Cyclosporine therapy was effective in reducing the ocular and lacrimal gland disease. Intraocular inflammation was present in 73% of control animals but in only 15% of cyclosporine-treated animals (P < 0.003). Multifocal lacrimal gland inflammatory infiltrates were present in 100% of controls but in only 23% of cyclosporine-treated animals (P < 0.0001). Mean percent area involved by lacrimal gland inflammation was reduced from 19.7% to 4.7% by cyclosporine therapy (P = 0.0003). Systemic autoimmune disease manifestations, including lymphoproliferation, vasculitis, glomerulonephritis, and serologic abnormalities, also were improved. CONCLUSIONS: Chronic cyclosporine therapy, started at an early age, is effective in controlling the autoimmune disease in MRL/lpr mice, including the ocular and lacrimal gland lesions.

Paradoxic effect of anti-CD4 therapy on lacrimal gland disease in MRL/Mp-lpr/lpr mice.

PURPOSE: MRL/Mp-lpr/lpr mice (MRL/lpr) spontaneously develop lacrimal gland inflammatory lesions and are a model for the human disease Sjögren's syndrome. Therapy with monoclonal antibodies (mAb) to CD4 ameliorates the autoimmune renal, vasculitic, and intraocular inflammatory lesions in MRL/lpr mice. The effect of anti-CD4 mAb therapy on lacrimal gland immunopathology was evaluated. METHODS: From 1 to 5 months of age, MRL/lpr mice were treated with weekly intraperitoneal injections of 2 mg anti-CD4 mAb, after which they were killed and their lacrimal glands were removed for histologic evaluation and immunocytochemistry. Control mice were administered weekly intraperitoneal injections of either saline or normal rat immunoglobulin. RESULTS: Anti-CD4 mAb treatment produced no reduction in lacrimal gland inflammation but did change its morphology. In control mice, there were multiple sharply delineated foci of inflammatory cells in the lacrimal gland, whereas in anti-CD4 mAb-treated mice, there was a more diffuse inflammation surrounding ill-defined foci that spread throughout the gland. Immunocytochemistry revealed that in control mice, lesions were composed predominantly of CD4+ T cells, but in anti-CD4 mAb-treated mice, CD8+ T cells predominated. CONCLUSIONS: Although anti-CD4 mAb therapy of MRL/lpr mice eliminated autoimmune renal disease, autoantibody formation, and ocular inflammatory disease, it had a paradoxic effect on lacrimal gland lesions. Lacrimal gland lesions in the anti-CD4 mAb-treated mice were not decreased, but they had a different morphology and a different immunocytochemical profile.

Murine model of ocular infection by a human biovar of Chlamydia trachomatis.

PURPOSE: A human biovar of Chlamydia trachomatis was used to develop a murine model of ocular chlamydial infection. The inbred mouse model will allow detailed immunologic studies during ocular infection, and use of a human biovar for infection may aid in identification of appropriate vaccine strategies against chlamydial infections. METHODS: BALB/c, C3H/HeN, and C57B1/6J mice (n = 5 to 10 mice/group) were topically infected in the conjunctiva with C serovar of C. trachomatis. The effects were tested of single and repeated infection with 5000 inclusion-forming units (IFU) in 5 microliters and different inoculum doses. Conjunctival surfaces of both eyes were swabbed for microbiologic signs (isolation culture or direct fluorescent antibody staining) of infection over 4 to 6 weeks. Conjunctivae were removed for histopathologic study, and lymphocytes from draining cervical lymph nodes and spleens were tested for chlamydia-specific proliferative responses. Serum was obtained from all mice and tested for anti-chlamydial antibodies. RESULTS: BALB/c and C3H/HeN mice developed dose-dependent microbiologic, histopathologic, and immunologic evidence of ocular infection. Eyes of mice were culture-positive from day 7 through at least day 21, with the peak of infection at days 10 to 14 after infection. Histopathologically, the development of conjunctival subepithelial mononuclear infiltration, exudate, and loss of goblet cells occurred within 1 week. Dose-dependent lymphoproliferative responses to whole chlamydial elementary bodies were observed; anti-chlamydial antibody was detected by immunoblotting only in infected mice. CONCLUSIONS: Several strains of inbred mice are susceptible to human chlamydial biovars and may provide a useful alternative disease model in which to study the immunopathogenesis of ocular chlamydial infection and test of vaccine candidates derived from clinically relevant human biovars.

Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing.

Duchenne muscular dystrophy (DMD) is characterized by clinical weakness and progressive necrosis of striated muscle as a consequence of dystrophin deficiency. While all skeletal muscle groups are thought to be affected, enigmatically, the extraocular muscles (EOM) appear clinically unaffected. Here we show that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium ([Ca2+]i) in EOM. At variance with a previous report, we find no evidence for dystrophin-related protein/utrophin up-regulation in EOM. In vitro experiments demonstrate that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated [Ca2+]i levels when compared with pectoral musculature. We believe that EOM are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. Our results indicate that modulating levels of [Ca2+]i in muscle may be of potential therapeutic use in DMD.

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex.

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

A chlamydial major outer membrane protein extract as a trachoma vaccine candidate.

PURPOSE: As shown in infected humans and in animal models of chlamydial infection, the major outer membrane protein (MOMP) of Chlamydia trachomatis is immunogenically potent. The purpose of this investigation was to test in the cynomolgus monkey model of trachoma a new extract of MOMP as a candidate vaccine against ocular chlamydial infection. METHOD: The nonionic detergent octyl-beta-D glucopyranoside (OGP) was used to extract MOMP from purified C. trachomatis (serovar C) elementary bodies. Protective immunization with OGP-MOMP by mucosal and systemic routes was compared in the cynomolgus monkey model of trachoma. All control and immunized monkeys were challenged by topical application of infectious C. trachomatis to the conjunctivae 35 days after the initiation of immunization. RESULTS: Immunization with OGP-extracted MOMP successfully induced chlamydia-specific local and systemic immunity to MOMP and to whole organism before challenge and early clearance of infection by systemically immunized monkeys. Although ocular disease was not significantly reduced in either immunized group compared to control animals, the lowest clinical and microbiologic disease scores developed in two animals in the mucosal group with the highest immunoglobulin A tear antibody titers at day 0 to 14, whereas higher tear and serum immunoglobulin G correlated with reduced disease in the systemically immunized group. CONCLUSIONS: These data demonstrate that despite evidence of vigorous MOMP-specific and other chlamydia-specific serologic and cell-mediated immunity, as well as anamnestic serologic responses to chlamydia, vaccination with OGP-MOMP was only partially protective against chlamydial ocular disease. The partial protection correlated best with tear immunoglobulin A responses after mucosal immunization and with local and systemic immunoglobulin G responses after peripheral immunization, suggesting that alternative chlamydial antigens may have to be considered in future vaccine development to induce more effective protective immunity and that evaluation of efficacy must be appropriate to route of immunization.

Sea star factor blocks development of T-dependent antibody secreting clones by preventing lymphokine secretion.

Sea star factor (SSF), a protein of 39 kDa purified from macrophage-like coelomocytes of the echinoderm Asterias forbesi, has potent immunosuppressive effects on T-dependent but not T-independent antibody responses in vivo. SSF at a concentration of 0.5 microgram/ml markedly inhibits T-dependent antibody production in vitro by fluorescein (Flu)-specific B cells responding in clonal microculture to antigenic stimulation with Flu-conalbumin via the conalbumin-specific T cells D10.G4.1 (D10). At this concentration of SSF, Ig secretion induced by a T cell-independent stimulus, lipopolysaccharide (LPS), is not affected. Inhibition of antibody production in T-dependent microcultures by SSF can be completely overcome in a dose-dependent fashion by addition of lymphokine-rich supernatants from stimulated cultures of D10 cells. The possibility that SSF suppresses production of requisite cytokine growth factors from T cells was substantiated by the finding that SSF diminishes concentrations of stimulatory cytokines detectable in supernatants from antigen-stimulated cultures. Nevertheless, levels of intracytoplasmic mRNA for IL-4 and IL-5 are not detectably altered by concentrations of SSF that suppress antibody production. Furthermore, when cultures of D10 cells stimulated in the presence of SSF are subjected to freezing and thawing to release intracytoplasmic lymphokines, total levels of stimulatory cytokines are not lower than those in cultures without SSF. These results suggest that SSF inhibits antibody responses by limiting the availability of lymphokines produced by helper T cells. The mechanism for this inhibition may involve either direct effects of SSF on T cells or a block in effective T cell-B cell interaction.

Effects of early and late treatment with anti-CD4 monoclonal antibody on autoimmune disease in MRL/MP-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation, with target organ inflammatory lesions composed largely of CD4+ (helper) T cells. Previous reports have demonstrated that anti-CD4 monoclonal antibody (mAb) treatment of MRL/lpr mice from 1 to 5 months of age resulted in a dramatic reduction in both the frequency and the severity of autoimmune disease. In order to investigate the effects of early, short-course and late, short-course anti-CD4 mAb therapy on the autoimmune disease in MRL/lpr mice, groups of 12 to 15 animals were treated with weekly intraperitoneal injections according to one of four regimens: (i) anti-CD4 mAb from age 1 to 5 months (continuous treatment); (ii) anti-CD4 mAb from age 1 to 3 months (early treatment); (iii) anti-CD4 mAb from age 3 to 5 months (late treatment); and (iv) either normal saline or rat immunoglobulin (control treatment). Continuous treatment resulted in a dramatic reduction of both frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Early treatment also resulted in a significant reduction in autoimmune disease, while late treatment had little effect. Glomerulonephritis was detected in none of the animals in the continuously treated group (P < 0.05), 38% of those in the early-treated group (P = < 0.05), 92% of the late-treated group, and 100% of controls. The titer of antinuclear antibodies, of anti-dsDNA antibodies, and total immunoglobulin levels were all significantly reduced in the continuous-treatment and early-treatment groups, but not in the late-treatment group. Murine antibodies to rat anti-CD4 mAb were present in the late-treatment group. These results indicate that early short-course anti-CD4 mAb treatment of MRL/lpr mice is effective in ameliorating the autoimmune disease in this model, while late-treatment is ineffective, probably due to the induction of antibody directed against anti-CD4 mAb itself.

Murine models of Sjögren's syndrome.

Autoimmune MRL/lpr, MRL/+, and NZB/W mice all develop lacrimal gland inflammatory lesions, which consist of focal mononuclear inflammatory cell infiltrates. Each strain has a different immunocytochemical profile, which appears to be related to the underlying immunologic defects present in that mouse. The appearance of these lesions parallels the evolution of the systemic autoimmune disease. The lesions are dynamic over time with the early appearance of CD4+ T cells (helper T cells) for each strain. Subsequently, there is an accumulation of B cells over time in MRL/+ and NZB/W mice. In the two more rapidly evolving mouse models, MRL/lpr and NZB/W, there is a progressive decline in the percentage of CD8+ cells. Conversely, in the slowly evolving MRL/+ lacrimal gland lesions, there is a persistent and unchanging percentage of CD8+ T cells (suppressor/cytotoxic T cells). Autoimmune mice provide models for the human disorder Sjögren's syndrome and a mechanism for better understanding the immunopathogenesis of autoimmune lacrimal gland disease.

Intraocular endometrium in the rabbit as a model for endometriosis.

The model described suggests that endometrium can be successfully transplanted to the rabbit eye and observed through a slit lamp for morphological changes such as vascularization. Sampling of aqueous humor in volumes adequate for biochemical measurements have been demonstrated. Autografts of rabbit endometrium survived for up to 181 days. Although xenografts of human endometrial and endometriotic tissue demonstrate some adherence and vascularization, there is indication of immune rejection by day 7. Other treatment regimens will be explored with the objective of prolonging the graft survival time.

Frequency of antigen-specific B cells during experimental ocular Chlamydia trachomatis infection.

Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cervical lymph nodes by an ELISPOT assay in a cynomolgus monkey model of trachoma. These local sites contained numbers of chlamydia-specific B cells that were higher than those in distant inguinal lymph nodes and peripheral blood. The numbers of chlamydia-specific immunoglobulin G-secreting B cells observed were 5 to 57 per 10(6) cells in conjunctiva and 24 to 996 per 10(6) cells in cervical lymph nodes during conjunctival infection or after challenge of immune monkeys with the chlamydial 57-kDa heat shock protein (hsp60). These studies demonstrate a large chlamydia-specific B-cell component in the conjunctiva during ocular chlamydial infection. These results are similar to our findings for chlamydia-specific T-cell responses.

Anti-CD4 monoclonal antibody therapy suppresses autoimmune disease in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ inflammatory lesions are composed largely of CD4+ "helper" T cells, while the massively enlarged lymph nodes are composed primarily of CD3+ CD4- CD8- TCR alpha/beta + "double-negative" T cells. In this study we investigated the effect of treatment of MRL/lpr mice with anti-CD4 monoclonal antibody (mAb); control groups consisted of animals treated with normal saline or rat immunoglobulin (Ig). Anti-CD4 mAb treatment, which was started at 4 weeks and continued through 20 weeks of age, resulted in a dramatic reduction of both the frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Anti-CD4 mAb therapy markedly reduced the frequency of glomerulonephritis and eliminated vasculitis of the major renal arterial branches. Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Ig-treated, but in only 1 of 9 anti-CD4 mAb-treated mice; vasculitis was detected in 6 of 9 saline-treated, 7 of 9 rat Ig-treated, but in none of 9 anti-CD4 mAb-treated mice. The frequency of antinuclear antibodies, titer of anti-dsDNA antibodies, and total Ig levels were all significantly reduced by anti-CD4 mAb therapy. These data support the hypothesis that CD4+ T cells play a central role in the disease process in this autoimmune strain.