Adjuvant-induced arthritis in four inbred strains of rats. An in vitro study of peripheral T and B lymphocytes

The lymphoblastic response (LTT) to non-specific mitogens (PHA, PWM and CoA) of peripheral lymphocytes was investigated at days 0, 7, 14, 21 and 28 after adjuvant injection in four strains of inbred rats: Wistar (WAG), Long Evans (LE), Lewis (LEW) and Brown Norway (BN). LTT was assessed by using 18 hours H³ TdR incorporation in 5 days cultures of whole blood (micromethod).The statistical treatment of data, using principal components multifactorial analysis and analysis of variance showed a striking difference between strains.In control animals the responses to PHA and PWM were correlated and were higher in LE and WAG than in LEW and BN (BN=LEW相似文献   

Hapten antibody production and the relevance of allogeneic reactions to elimination of the carrier effect

Lewis (LEW) rats immunized 3 weeks before by injection of DNP-KLH together with Bordetella pertussis showed high levels of DNP antibody as judged by serum binding of 10(-7) M 3H-DNP-lysine 10 days after secondary immunization with DNP-KLH. Sera obtained from LEW rats following secondary immunization with DNP-BGG showed reduced DNP hapten binding. However, injection of 10(8) F1 hybrid Lewis X Brown Norway spleen cells into DNP-primed LEW rats 2 days before secondary immunization with DNP-BGG significantly increased the level of serum binding of 3H-DNP-lysine. These results provide evidence that the allogeneic cellular reaction associated with a host-versus-graft response induced by injection of F1 hybrid lymphoid cells into DNP-primed parental strain recipients partially obviates the requirement for carrier-specific T cells in the secondary anti-DNP response thereby providing a stimulus for triggering primed host B cells to produce antibody.     

Regulation of resistance against adjuvant arthritis in the Fisher rat.

Inbred female Lewis (LEW/N) rats develop a severe chronic arthritis in the adjuvant arthritis (AA) model, histocompatible Fisher (F344/N) rats are resistant and germ-free Fishers (GF F344) are again susceptible. In this study we show that the F344 rat can become susceptible to AA, using Mycobacterium tuberculosis (M.tb.) in the powerful adjuvant paraffin oil, instead of mineral oil (Freund's incomplete adjuvant (FIA)). This indicates that the F344 rat does not lack T effector cells. To examine further mechanisms responsible for suppression, we determined the level of plasma corticosterone in response to IL-1 alpha in Lewis, F344 and GF F344 rats. IL-1 alpha induced only low amounts of corticosterone in Lewis rats, but high amounts in both F344 and GF F344 rats. The GF F344 rats are susceptible to AA, but the severity of the disease is reduced compared with Lewis rats. This indicates that corticosterone may be an important mechanism to suppress disease development, but not the only mechanism. In addition we investigated whether T suppressor cells play a role in the resistance of the F344 strain. This was performed by pretreating the animals with the immunomodulating drugs cyclophosphamide (Cy) and cyclosporin A (CsA). We were unable to make the F344 rat susceptible to AA, indicating that active suppression does not play a role in the induction phase of arthritis. This finding is confirmed in adoptive transfer experiments of AA from Lewis to F344 rats. Our data suggest the lack of a strong pre-existing suppression in the F344 rats, and indicate that suppression is generated upon bacterial challenge. Whether suppression is overruled probably depends on the power of adjuvants used and potential control by corticosteroids.     

A Rad LV-Induced γδ T Cell Lymphoma Displaying an Antitumoral Cytotoxicity

Brown Norway (BN) rats are poor responders to T-cell mitogens and alloantigens when compared to Lewis (LEW) rats. This is dependent partly upon a defect in IL-2 production. The TH2-mediated immune abnormalities observed in BN rats injected with mercuric chloride (HgCl₂) are self-limited and it is probable that this regulation phase involves TH1 -like cells. This paper reports on a study of the ability of lymph node cells (LNC) from normal BN and LEW rats and from HgCl₂-injected BN rats to produce IL-2 and to proliferate when stimulated in vitro by Con A or alloantigens in mixed lymphocyte reaction (MLR), as well as to develop a cytotoxic T lymphocyte (CTL) response to alloantigens. This study will confirm that LNC from BN rats proliferate less than LNC from LEW rats, that the former produce less IL-2 than the latter, and that the proliferative response is restored partially after addition of IL-2. In addition, it is shown (1) that the CTL response is defective in normal BN rats when compared to that of normal LEW rats, and (2) that, after the second week of HgCl₂ injections, the proliferative responses to Con A and alloantigens are improved as well as IL-2 production, and a complete restoration of CTL function is observed. These results show that normal BN rats are deficient in the induction of THl-like cells and that, from the second week of HgCl₂ injections, these TH1 functions improve.     

Haplotype-specific suppressor T cells mediating linked suppression of immune responses elicited by third-party H-2 alloantigens

Specific suppressor T cells (Ts) were induced in vitro by incubation of mouse spleen/lymph node cells with allogeneic heat-treated cells. These Ts inhibit mixed lymphocyte reaction (MLR) in a haplotype-specific manner. Ts also suppress cell proliferation induced by third-party H-2 alloantigens provided these are expressed on the same cell surface as at least some of the H-2 antigens used for Ts activation. Ts activated by H-2 plus non-H-2 alloantigens suppress an MLR induced by irrelevant H-2 alloantigens if these are expressed on the same cell surface as the non-H-2 alloantigens used for Ts activation. Products of the H-2 region or non-H-2 alloantigens which are not able to stimulate cell proliferation do not activate Ts. These Ts are first demonstrable after 4 days of incubation of lymphoid cells with heat-treated allogeneic cells and they inhibit MLR only if added at the very beginning of the culture. Exogenous interleukin 2 does not overcome suppression and the suppression is not due to a cytotoxic effect, since heat-treated cells do not elicit cell proliferation or cytotoxic cells. Moreover, the specific Ts differ in their Thy-1+,Ly-1+,2-phenotype from Ly-2+ allospecific cytotoxic cells. Thus specific Ts could be induced in vitro, which demonstrate linked suppression for third-party H-2 alloantigens provided these are expressed on the same cell surface as the antigens used for Ts activation.     

Immunosuppressive macrophages induced by arthropathic peptidoglycan-polysaccharide polymers from bacterial cell walls.

Rats injected with peptidoglycan-polysaccharide polymers derived from group A streptococcal cell walls (PG-APS) develop a chronic, remittant, erosive synovitis. Spleen cells from injected rats failed to proliferate when stimulated in vitro by Con A or PHA, unless nylon wool adherent cells were first removed. The suppression could also be reversed by removing phagocytic cells which had ingested carbonyl iron. Cells from control rats were suppressed in vitro by co-culture with unfractionated or nylon wool-adherent cells from PG-APS injected rats, and the suppressor activity was still expressed after exposure of the suppressor cells to 3,000 rad of irradiation. Addition of catalase and indomethacin to cultures only partially reversed the suppression. T lymphocytes from rats given a single arthropathic dose of PG-APS remained suppressed for at least 86 days after injection. Cells from rats given a low, non-arthropathic dose of PG-APS did not become suppressed. Cells from the Buffalo rat, which is resistant to development of PG-APS-induced chronic arthritis, showed less suppression than cells from the susceptible Lewis and Sprague-Dawley rat strains.     

THE REGULATORY ROLE OF I-J SUBREGION IN NEONATAL TOLERANCE INDUCTION TO H-2D ALLOANTIGENS

The mechanism of neonatally induced transplantation tolerance was studied in two mouse strain combinations involving differences at the D region of the H-2 complex only or at the same D region plus I-J subregion (including I-E, I-C, S and G regions). In the strain combination with the H-2D difference only, cells from tolerant mice proliferated markedly in the MLR assay when incubated with antigens tolerated in vivo, whereas the MLR reactions were negative in the combination with D plus I-J region disparities. In the latter combination cells from tolerant mice also did not respond to third-party antigens and their incubation with the tolerated antigens led to the suppression of cell proliferation. This non-specific suppression was absent in cells from tolerant mice in the strain combination, which differed in I-C, S, G and D alloantigens. Specific suppressor cells, which inhibited the development of cytotoxic cells, were demonstrated in tolerant mice of both strain combinations. The results show that, in addition to the specific suppressor cells induced by H-2K or H-2D alloantigens, non-specific suppressor cells induced by the I-J region disparity that may regulate the resultant activity against H-2D (and probably also H-2K) alloantigens are involved in transplantation tolerance.     

Antigen specific down regulation of murine collagen induced arthritis: T suppressor cell circuits in arthritis immunotherapy

The present article summarizes a series of experiments which have been performed to describe an antigen-specific suppressor cell pathway for the suppression of the erythema and edema associated with an animal model of rheumatoid arthritis, collagen induced arthritis (CIA). Initial studies utilized the adoptive transfer of splenic cell subpopulations to establish the presence of suppressor cells in lymphoid tissues of mice which were suppressed for collagen induced arthritis. Subsequent studies generated T cell hybridomas from animals which had been suppressed for collagen induced arthritis by a single injection of a large quantity of Type II collagen. The T cell hybridomas varied in their self surface expression of glycoproteins which are associated with genetically determined functions. The suppressor T cells generated, described a regulatory suppressor cell pathway comprised of at least afferent suppressor T cells and effector suppressor T cells. The cells act in an antigen-specific fashion with regard to the suppression of collagen induced arthritis but appear to be polymorphic in their recognition of the interstitial collagens. The studies, taken together, indicate that the use of antigen specific T suppressor cells in the form of T cell hybridomas can be utilized as a form of immunotherapy in experimental arthritis.     

BCG-induced suppressor T cells optimal conditions for in vitro induction and mode of action.

In vitro activation with BCG of T cells from healthy individuals vaccinated with BCG lead to the induction of suppressor cells that suppressed the proliferation of fresh T cells in response to specific antigen. Kinetics of their induction revealed that they became radioresistant by day 8 and persisted up to 18 days of the culture period. Optimal antigen and monocyte concentrations as assessed by proliferation during the induction phase also resulted in maximum suppression. The strongest suppressor activity was observed when suppressor cells were added at an early time of fresh cell activation. IL-1 production from adherent cells in response to BCG was not affected, but, IL-2 production by T-cells was considerably reduced in the presence of suppressor cells. IL-1 containing supernatants and affinity purified IL-1 exogenously added to the culture system did not affect suppression. Whereas, recombinant IL-2 partially abrogated suppression in a dose-dependent manner. Further experiments suggested that suppressor cells might have inhibited BCG induced IL-2 receptor expression on fresh T cells.     

Inhibitory effects of interferon-gamma on the T suppressor cell circuit in contact sensitivity

The effects of a partially purified, splenocyte-derived murine interferon (MuIFN-gamma N) and a recombinant IFN-gamma (MuIFN-gamma R) on the T suppressor pathway and on the T effector cells of delayed type hypersensitivity were investigated in a 2,4-dinitrofluorobenzene contact sensitivity model. Various T cell subpopulations, suppressor T cells of afferent and efferent types, and an auxiliary T suppressor cells as well as a T effector cell of delayed type hypersensitivity were induced and the functions assessed in transfer experiments. Confirming the results of earlier experiments obtained with IFN-alpha, beta, the MuIFN-gamma N preparation and the rec. MuIFN-gamma R: enhanced the decreased response in animals sensitized with an antigen overload to an optimal response; inhibited the afferent-acting T suppressor cell in vivo and in vitro; inhibited the Ts-eff response; blocked the auxiliary T suppressor cell response after intravenous injection to recipients of Ts-eff cells on day 0 and 1; and did not suppress the activity of the T effector cell of delayed type hypersensitivity in vivo and in vitro (the MuIFN-gamma R was not tested). We conclude that IFN-gamma preferentially inhibited the T suppressor cell circuit of contact allergy. These results are similar to our observations on the inhibitory effects of a pure interferon-alpha, beta on the regulatory T suppressor cell circuit in contact allergy. Selective suppression of different T subpopulations by IFN-gamma may be an important regulatory mechanism in delayed type hypersensitivity.     

A human autoantibody to renal collecting duct cells associated with thyroid and gastric autoimmunity and possibly renal tubular acidosis

Peripheral blood mononuclear cells (PBMC), obtained from BCG vaccinated healthy donors, were induced to proliferation by BCG for five days in vitro. When re-exposed to BCG, they failed to proliferate. However, they partially retained the ability to respond to Con A and allogeneic cells. The addition of graded numbers of such cultured cells to fresh autologous PBMC suppressed their proliferative response to BCG. These suppressor cells could also inhibit the proliferation of fresh cells to other mycobacterial antigens, both in particulate form, i.e. Mycobacterium leprae, or in soluble form, i.e. PPD and SPA30. However, these pre-cultured cells did not inhibit the response of fresh cells to non-specific mitogens, i.e. Con A and alloantigens. The inhibition of the response to non-mycobacterial soluble antigens, i.e. tetanus toxoid (TT) and diphtheria toxoid (DT) varied with little suppression in some individuals and stronger suppression in others. The suppression to BCG was found to be mediated by T cells. Subfractionation of T cells by monoclonal antibodies OKT4 and OKT8 allocated the suppressor cells to the OKT4+ class of T cells. The suppression in the autologous system was quite strong, whereas it was much weaker in allogeneic systems.     

Regulation of autoimmune arthritis by the pro-inflammatory cytokine interferon-gamma

The pathogenesis of T cell-mediated diseases like rheumatoid arthritis (RA) has typically been explained in the context of the Th1-Th2 paradigm: the initiation/propagation by pro-inflammatory cytokines, and downregulation by Th2 cytokines. However, in our study based on the adjuvant-induced arthritis (AA) model of RA, we observed that Lewis (LEW) (RT.1(l)) rats at the recovery phase of AA showed the highest level of IFN-gamma in recall response to mycobacterial heat-shock protein 65 (Bhsp65), whereas AA-resistant Wistar-Kyoto (WKY) (RT.1(l)) rats secreted high levels of IFN-gamma much earlier following disease induction. However, no significant secretion of IL-10 or TGF-beta was observed in either strain. Furthermore, pre-treatment of LEW rats with a peptide of self (rat) hsp65 (R465), which induced T cells secreting predominantly IFN-gamma, afforded protection against AA and decreased IL-17 expression by the arthritogenic epitope-restimulated T cells. These results provide a novel perspective on the pathogenesis of autoimmune arthritis.     

Resistance to the induction of EAE in AO rats: its prevention by the pre-treatment with cyclophosphamide or low dose of irradiation

Susceptibility to the induction of EAE was compared in AO, DA and Lewis strain of rats. As evaluated by clinical and histological criteria, AO rats exhibited significantly lower susceptibility to EAE induced with guinea-pig spinal cord (GPSC) tissue and complete resistance to the encephalitogenic challenge with rat myelin basic protein (BP) irrespective of antigen dose and adjuvant used. AO rats pre-treated with BP + Freund's incomplete adjuvant became completely unresponsive to the induction of EAE with GPSC + Freund's complete adjuvant (FCA) indicating that they do possess cells sensitive to some antigenic determinants of rat BP. In order to test whether the resistance to EAE is due to an active suppression, low dose of irradiation (300 rad) and cyclophosphamide (20 mg/kg) was applied prior to the induction of EAE. Selective depletion of radiosensitive cells facilitated the induction of EAE. Similarly, cyclophosphamide given 2 days prior to BP + FCA completely abrogated the resistance to EAE induction. Thus, it appears that the inability of BP + FCA to produce EAE in AO rats is due to the disproportionate activation of suppressor cells.     

Selective immunosuppression with anti-interleukin 2 receptor-targeted therapy: helper and suppressor cell activity in rat recipients of cardiac allografts

(LEW X BN)F1 cardiac allografts are rejected within 8 days in unmodified LEW rats. ART18, a mouse anti-rat IgG1 monoclonal antibody which binds specifically in vitro to the interleukin 2 receptor (IL 2R) molecule expressed primarily on activated T cells, prolongs allograft survival in a dose-dependent fashion to ca. 3 weeks (p less than 0.001) after being administered for 10 days after transplantation. This effect was related to the specificity of the antibody for IL 2R, as therapy with ART62 (a monoclonal antibody recognizing MHC class I antigen but not binding the rat IL 2R) was ineffectual. Suppressor activity was detected in spleen cells of ART18-treated grafted hosts: in vivo, splenic T suppressor/cytotoxic fraction adoptively transferred into normal LEW improved donor-specific but not third-party test graft survival (17 days, vs. 8 days, respectively, p less than 0.001); in vitro, mixed lymphocyte reaction was profoundly but nonspecifically inhibited (less than 5% of test mixed lymphocyte reaction, p less than 0.001 as compared to acutely rejecting controls). In contrast, splenic T helper (Th) cells from ART18-treated hosts were functionally depressed, as noted by their passive transfer into immunologically anergic B recipients of cardiac allografts (rejection in ca. 40 days, vs. ca. 13 days after transfer of Th from specifically sensitized rats). ART18 treatment also resulted in diminished elaboration of IL 2 as compared to normal (p less than 0.005) or acutely rejecting hosts (p less than 0.001); however, a remarkable increase in the production of IL 3 occurred (p less than 0.001). These results demonstrate that IL 2R-targeted therapy of immunocompetent graft recipients produces a selective immune defect in which donor-specific T suppressor cells are spared, but Th cells attenuated or destroyed. Decreased elaboration of IL2 concomitantly augments the release of IL 3, a lymphokine which might play a role in suppressor effect in vivo. In addition, IL 2R-targeted therapy of the immunodeficient graft recipients abrogates the capacity of alloactivated T cells to re-establish acute immune responsiveness.     

Role of CD8+ T Cells in Mercury-Induced Autoimmunity or Immunosuppression in the Rat

In Brown-Norway (BN) rats mercuric chloride induces an autoimmune disease characterized by an increase in serum IgE concentration, and by the production of anti-glomerular basement membrane antibodies responsible for a glomerulonephritis with a heavy proteinuria. (i) This disease results from a B-cell polyclonal activation probably due to frequent anti-class II T cells. (ii) The self limitation observed in this model is associated with both a decrease in the frequency of anti-class II T cells and the emergence of CD8+ T cells able to suppress these autoreactive T cells. (iii) In Lewis (LEW) rats which do not develop autoimmunity, HgC12 provokes the appearance of non-antigen-specific CD8+ T cells responsible for a depression of T-cell functions. The aim of this work was to test the effect of treatment with an anti-CD8 monoclonal antibody (MoAb) in both BN and LEW rates, Anti-CD8 MoAb-treated rats were effectively depleted in CD8+ T cells. However, neither the induction nor regulation phases of mercury-induced autoimmunity were modified in BN rats. Mercury-induced immunosuppression in LEW rats was abrogated; however, depletion in CD8+ T cells did not allow the disease to occur in that strain. Finally, CD8 depletion induced in normal BN rats rats the appearance of rare anti-class II T cells showing that these cells are normally present in that strain but negatively controlled by suppressor T cells.     

Delayed-type hypersensitivity to allogeneic mouse epidermal cell antigens. III. Analysis of suppressor cells

The specificity of suppressor T cells (Ts cells) induced by intravenous administration of allogeneic spleen cells was studied in mice using a delayed-type hypersensitivity (DTH) assay. The DTH responses were induced by subcutaneous injection of allogeneic epidermal cells (ECs) and were assayed by footpad swelling. Afferent-phase Ts cells (Ts-aff cells) were transferred into the recipient mice before ECs immunization. Treatment with monoclonal anti-Thy-1.2 antibody and complement abolished the suppression by Ts-aff cells in the DTH response. The suppression induced by Ts-aff cells, however, was resistant to the treatment with monoclonal anti-I-A or anti-Lyt-2.2 antibody and complement. These results showed that Ts-aff cells were Lyt-2-, Ia- T cells. Efferent-phase Ts cells (Ts-eff cells) were transferred before challenge of the DTH assay. Phenotypic analysis of these Ts-eff cells showed them to be Lyt-2+, Ia- T cells. Studies using several strains of congenic mice revealed the antigen specificity of both Ts cell subsets. Adoptive transfer of Ts-eff cells required H-2 restriction, but Ts-aff cells did not. We also induced cognate suppression of the DTH responses to the alloantigens mediated by Ts-eff cells.     

Rats made congenic for Oia3 on chromosome 10 become susceptible to squalene-induced arthritis

Several quantitative trait loci (QTLs) regulating the risk of experimental arthritis have been identified by genome-wide linkage analyses, but only the MHC has thus far been reported to transfer arthritis susceptibility in congenic animals. We have produced a congenic strain for Oia3, a genetic factor originally identified as an oil-induced arthritis (OIA) QTL in arthritis-prone DA rats. A 46 cM telomeric region of chromosome 10 encompassing Oia3 was transferred from DA rats to MHC-identical but minutely arthritis-susceptible LEW.1AV1 rats by selective breeding. Arthritis development was provoked in Oia3-congenic rats by intradermal injection of different adjuvant oils. One successful arthritis trigger was squalene, which is approved for vaccinations in humans and has been implicated in Gulf War syndrome. The endogenous cholesterol precursor squalene induced T cell infiltration into joints and macroscopic arthritis in Oia3-congenic rats and DA rats, whereas LEW.1AV1 rats were almost resistant. Arthritis onset, approximately 14 days post-injection, coincided with arrested body-weight gain and increased plasma levels of the inflammation markers fibrinogen and alpha 1-acid glycoprotein. Congenic rats displayed intermediate phenotypes compared with the two parental strains, and similar to rheumatoid arthritis in humans, female preponderance was observed in Oia3-congenic rats. Finally, recombinant rat strains were constructed and were used to map a susceptibility gene(s) in females to a telomeric 4--19 cM Oia3 subregion. The experimental system described allows transformation of multifactorial arthritis susceptibility into dichotomous phenotypes.     

Spontaneous recovery from early glomerular inflammation is associated with resistance to anti-GBM glomerulonephritis: tolerance and autoimmune tissue injury

Different susceptibility to anti-GBM glomerulonephritis (GN) among animal strains has been reported. Using our rat model for T cell-mediated anti-GBM GN, this study initiated an investigation on the mechanism related with GN susceptibility. Anti-GBM GN was induced either through immunization with the nephritogenic T cell epitope pCol(28-40) from Col4alpha3NC1 or through the transfer of specific T cells. WKY rats were highly susceptible to GN while immuno-compatible LEW rats were GN-resistant. GN-resistance in LEW rats was not associated to the immune response to pCol(28-40). First, both strains mounted a Th1 T cell response to pCol(28-40) with identical specificities; transfer of T cells from LEW to WKY rats induced glomerular injury. Second, co-transfer of antibody from WKY to LEW failed to induce GN. Time-course studies revealed that LEW rats did develop T cell-mediated inflammation in glomeruli at early stages similar to WKY rats, as evidenced by histopathology, proteinuria, CD4(+) T cell infiltration in glomeruli, and glomerular expression of inflammatory molecules. However, glomerular inflammation in LEW rats was transient followed by a full recovery. Thus, GN-resistance in LEW rats was due to its ability to contain early T cell-mediated autoimmune glomerular damage. Our model may reveal a potential tolerance mechanism after autoimmune tissue damage has been initiated.     

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.