An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen.

Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.

Abdominal intratesticular islet-xenograft survival in rats.

We investigated the survival of islet xenografts in the abdominal testes of normal, diabetes-resistant ACl and Wistar-Lewis (W-L) and in diabetes-prone BB/Wor rats. Islets were isolated from hamster donors, and after a period in tissue culture, they were injected into the abdominal testes of diabetic rats. Postoperatively, the rats were divided into two treatment groups. Four ACl rats received antilymphocyte serum (ALS) injections over a period of 30 days, and 13 ACl, 15 W-L, and 13 BB/Wor rats were given cyclosporin (CsA) according to the following regimen: 25 mg/kg i.p. for 7 days, then 7 mg/kg i.p. for 23 days. On day 30, immunosuppression was stopped. The results showed that none of the ALS-treated ACl rats remained normoglycemic for greater than 9 days. However, CsA therapy resulted in an extended mean duration of normoglycemia in the ACl and W-L rats of 160.0 +/- 36.0 and 131.0 +/- 31 days, respectively. By contrast, the mean duration of normoglycemia in the BB/Wor rats was 33.0 +/- 4.0 days. Furthermore, all of the BB/Wor rats reverted to diabetes after CsA was stopped. Therefore, the concomitant administration of an antigenic islet xenograft with CsA led to a state of unresponsiveness only in diabetes-resistant but not in diabetes-prone rats. Because the BB/Wor rats have demonstrable T-lymphocyte dysfunction, they may be unable to generate the CsA-induced suppressor T-lymphocytes required for the long-term acceptance of the graft by the host.

Islet allografts in the cryptorchid testes of spontaneously diabetic BB/Wor dp rats: response to glucose, glipizide, and arginine.

The aim of this study was to examine insulin and glucagon secretory patterns in successfully transplanted spontaneously diabetic BB/Wor dp rats. Diabetic, BB/Wor dp rats received abdominal, intratesticular islet grafts of MHC-compatible BB/Wor dr donor rats without immunosuppression. After a period of 74 +/- 15 days of normoglycemia, they were given the following challenges: 1) glucose, by mouth, 2) a single oral dose of glipizide, with glucose, and 3) arginine, by iv infusion. The pertinent results included the mean fasting plasma glucose levels of control, Sprague-Dawley (C), of transplanted BB/Wor dp (T), and nontransplanted, insulin treated, diabetic BB/Wor dp (D), and they were, respectively, 97 +/- 4 mg/dl, 110 +/- 3 mg/dl, and 350 +/- 40 mg/dl. Fasting plasma insulin levels in C and T rats were 21.9 +/- 3 microU/ml, and 20.4 +/- 2 microU/ml, respectively. Fasting plasma glucagon levels in C, T, and D, were 37.8 +/- 5.7 pg/ml, 43.4 +/- 4.6 pg/ml, and 47.4 +/- 4.9 pg/ml, respectively. During oral glucose tolerance test, the pattern of insulin secretion in the C and T rats was identical with a peak attained at 15 min. Glucose caused a 70% suppression of plasma glucagon levels in C rats (P less than 0.01); T rats suppressed 14%, but this was not statistically significant; D rats failed to suppress. Glipizide plus glucose caused an improved glucose tolerance in T rats without significantly affecting insulin levels. In the same rats, glipizide resulted in a significant suppression of glucagon compared with levels in the presence of glucose alone. Arginine caused a minimal release of insulin in T rats and a major glucagon secretory response in D rats. Pancreatic glucagon content was significantly (P less than 0.03) lower in C and T, compared with D rats. Furthermore, the transplanted testes of T contained substantial amounts of glucagon. In summary, these data suggest that grafted testes in spontaneously diabetic BB/Wor dp rats contain both beta and alpha-cells and that these cells have the capacity to respond to specific secretagogues independently.

5-Azacytidine increases the synthesis of embryonic hemoglobin (E2) in murine erythroleukemic cells.

The addition of 5-azacytidine to erythroleukemic cells which were induced to differentiate with DMSO or BA altered the expression of the hemoglobins. After the addition of 5-azacytidine there was an increase in hemoglobin synthesis especially in the embryonic E2 band. The beta-globin increased in synthesis after 5-azacytidine treatment. The level of hemoglobin synthesis in DMSO-induced cells is less than BA-induced cells while the effect of the 5-azacytidine stimulation was greater with DMSO induction than with BA induction.

The effect of glutathione on HL-60 treated with dimethylsulfoxide, butyric acid or 12-O-tetradecanoylphorbol-13-acetate.

HL-60 promyelocytic leukemic cells can be induced to differentiate into granulocytes or macrophages. Reduced glutathione lyses undifferentiated HL-60 cells but has minimal effect on their differentiated counterparts. The addition of reduced glutathione to HL-60 promyelocytic leukemic cells retards cell growth and lyses cells. HL-60 cells can be induced to differentiate into granulocytes with dimethylsulfoxide butyric acid or into macrophages with 12-O-tetradecanoylphorbol-13-acetate. After treatment of HL-60 cells with these inducing agents the HL-60 cells become unresponsive to the effects of glutathione.

I-Aq and I-Ap bind and present similar antigenic peptides despite differing in their ability to mediate susceptibility to autoimmune arthritis.

Susceptibility to collagen induced arthritis (CIA) in the murine model is linked to expression of the MHC class II alleles, I-Aq and I-Ar. We have examined the molecular basis for this MHC-linked susceptibility by studying the antigen presentation function of two class II molecules, I-Aq and I-Ap, that are closely related yet differ in mediating susceptibility to CIA. These class II molecules differ by only 4 amino acids, yet only mice expressing I-Aq develop CIA. Although the I-Ap molecule can bind the same immunodominant determinant from type II collagen as I-Aq, H-2p APC have difficulty generating I-Ap:CII peptide complexes when processing of CII is required. Immunization of H-2p mice with type II collagen (CII) generated only a weak T cell response when compared to H-2q mice, whereas immunization with the a CII peptide containing the dominant determinant induced a strong T cell response in both strains. In antigen presentation assays, H-2p APC were very inefficient in stimulating T cells when native CII was used as antigen, however they presented CII synthetic peptides with similar efficiency as H-2q APC. Processing and presentation of other antigens by H-2p APC was not affected. Using soluble class II binding assays, the affinity of I-Ap for the CII dominant peptide was 10 to 50 fold lower than I-Aq, however, this reduced affinity was not a general defect in I-Ap function. I-Aq and I-Ap had virtually identical affinities for binding other antigenic peptides. These data indicate that MHC-based susceptibility to autoimmunity may involve more than simple determinant selection and that the successful generation of an antigenic peptide by processing may be related to the overall affinity of the peptide for the MHC molecule.

Intratesticular islet xenograft survival in relation to tissue cyclosporine levels.

A study of the comparative survival of islet xenografts using a combination of treatment modalities was carried out in the spontaneously diabetic BB/Wor rat. Islets were isolated from hamster donors, and after 4 to 6 days of incubation the cells were injected either into the immunologically privileged abdominal testis or into the nonimmunologically favored renal subcapsular space. Postoperatively the rats were given cyclosporine at two different dose schedules. The results showed that islets injected into the abdominal testis of nonimmunosuppressed rats caused the induction of normoglycemia with a mean duration of 8.3 +/- 1.3 days. A significant prolongation of normoglycemia to a mean of 36.1 +/- 4.5 days occurred in rats that were given abdominal, intratesticular islet xenografts and cyclosporine for 30 days. The longest average survival in excess of a mean of 90.1 +/- 6.5 days was achieved in rats that were given abdominal, intratesticular islet xenografts and cyclosporine continuously, every other day. All of the grafted rats reverted to diabetes upon the cessation of cyclosporine. A similar cyclosporine regimen failed to prolong islet xenograft survival for longer than a mean of 9.0 +/- 2.2 days in rats that were given islet xenografts injected into the renal subcapsular space. Extended survival of abdominal, intratesticular islet xenografts corresponded with trough plasma and testis cyclosporine levels of 457 +/- 46 ng/mL and 643 +/- 45 ng/g, of wet weight, respectively. It is concluded that islet xenografts are protected against immune destruction in the BB/Wor rat with type 1 diabetes only as long as the cells are injected into an immunologically privileged site and the host is continuously immunosuppressed with cyclosporine.

Glutathione mediated lysis of HL-60 cells.

Various concentrations of glutathione (GSH), between 50-2000 micrograms/ml, were shown to retard the proliferation of HL-60 cells, with the optimum level being 200 micrograms/ml. Using electronic cell volume, GSH was shown to increase the percentage of small size cells and debris, and decrease the population cell volume and the percentage of large size cells. This data combined with chromium-51 release, trypan blue exclusion, and morphology data suggested that GSH was lysing HL-60 cells. Catalase eliminated the GSH stimulated cell lysis suggesting a H2O2 mediated mechanism was involved.

Prolonged intratesticular islet allograft survival is not dependent on local steroidogenesis.

The mechanisms that control the privileged survival of intratesticular organ allografts are not known. It had been postulated that the elevated levels of testicular steroid hormones, testosterone and/or progesterone, could be responsible for the inhibition of the local immune response. The goals of this study were to examine intratesticular islet allograft survival in rats in which both germ cell and Leydig cell function had been selectively destroyed. A chemical castration was induced in diabetic Sprague-Dawley rats with the chronic administration of a GnRH analog, leuprolide. In addition, both germ cell function and steroidogenesis were severely impaired by means of the surgical removal of the pituitary in diabetic rats. Pancreatic islets were isolated from Wistar-Lewis rats and were then implanted into the abdominal testes of leuprolide-treated and of hypophysectomized rats. No immunosuppression was given to the grafted rats. The results showed that long-term allograft survival occurred in the abdominal testes deprived of germ cells, of testosterone and of progesterone.

Embryonic hemoglobins in mouse erythroleukemic cells.

Multiple hemoglobins are synthesized in the T3C12 murine erythroleukemic cells after induction with butyric acid and hemin. The pI values determined by IEF and globin composition of these hemoglobins suggests that A, E2, and E3 hemoglobins are synthesized in T3C12 cells. The embryonic mouse globins x, y, z and alpha from the yolk sac cells could be separated by acid urea triton x-100 electrophoresis. The mobility of the globins are the following alpha greater than z greater than y greater than x.

Differential hemoglobin synthesis in murine erythroleukemic cells: hemin effects.

The addition of butyric acid to murine erythroleukemic cells (clone T3Cl2) induced the cells to differentiate, producing adult hemoglobin (A, alpha 2,beta 2) and an embryonic hemoglobin (E2, alpha 2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four-fold and the synthesis of embryonic hemoglobin two-fold; the relative synthesis of the alpha and beta globins increased more than the y globin. The embryonic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.

Vaccination with a recombinant V alpha domain of a TCR prevents the development of collagen-induced arthritis.

A recombinant TCR domain, derived from a T cell hybridoma that recognizes an immunodominant type II collagen epitope, was used to vaccinate against collagen-induced arthritis in DBA/1 (H-2q) mice. The recombinant TCR domain comprises VA11.1-JA17 gene segments and is representative of the V alpha domains expressed by oligoclonal T cells in this disease model. Vaccination of mice 28 days before type II collagen (CII) immunization with this V alpha 11.1 domain resulted in a significantly decreased incidence of arthritis in DBA/1 mice, in contrast to vaccination with a V alpha 4-J alpha 40 domain derived from an encephalitogenic T cell hybridoma specific for MBP. Disease blockade is accompanied by a reduction in T and B cell responses to both the immunogen bovine CII and the autoantigen murine CII. V alpha 4 and V alpha 11.1 domains were found to be highly immunogenic in DBA/1 mice, inducing both T cell proliferation and the production of V alpha specific Abs, indicating that the vaccination effect of V alpha 11.1 is specific. This is the first report of V alpha-directed immunotherapy in an autoimmune disease model and demonstrates the potential use of recombinant TCR vaccines in the treatment of autoimmune diseases that involve oligoclonal autoreactive T cells.

Identification of MHC class II and TCR binding residues in the type II collagen immunodominant determinant mediating collagen-induced arthritis.

Collagen-induced arthritis (CIA), an autoimmune arthritis model, is elicited by the immunization of genetically susceptible strains of mice with type II collagen (CII). We have analyzed the molecular interactions that occur during the presentation of the immunodominant determinant within CII(257-270) by the murine class II susceptibility allele, I.Aq. Utilizing a soluble I-A binding assay and clonally distinct CII-specific T cells, we have identified the residues that control the ability of the CII(257-270) peptide to bind to I-Aq and those that interact with the TCR. In competitive binding assays with a panel of analog peptides, only two residues within CII(257-270) were found to participate in the binding of this peptide to I-Aq, residues 260 (Ile) and 263 (Phe). When these substitutions were combined into a single peptide, no binding of the peptide to I-Aq could be detected. Although no other substitutions decreased the binding affinity of the peptides, substitution of several amino acid residues lying outside of the determinant core increased the peptide's affinity for I-Aq and in some instances greatly enhanced the potency of the peptide in stimulating T cells. In antigen presentation assays, clonotypic variation in the recognition of several analog peptides indicated that residues 261, 262, 264, 266, and 267 are likely TCR contact sites. Since residue 266 interacts with the TCR and is the only residue in this determinant that differs between chick/bovine CII and mouse CII, these data indicate that immunity to the autoantigen may play a role in this model.

Characterization of signal transduction through the TCR-zeta chain following T cell stimulation with analogue peptides of type II collagen 260-267.

The immunodominant T cell determinant of type II collagen (CII) recognized by DBA/1 mice (I-Aq) is CII 260-267. The aims of this study were to determine the role of the amino acid residues within CII 245-270 in T cell signal transduction. To that end, we utilized I-Aq-restricted, CII-specific T cell hybridomas and examined tyrosine phosphorylation of TCR-zeta following stimulation with either wild-type CII 245-270 or a panel of analogue peptides. A variety of patterns occurred, ranging from increased phosphorylation of TCR-zeta to either partial or a complete abrogation of phosphorylation. Critical substitutions also completely abrogated the phosphorylation of ZAP70, a downstream molecule in TCR-zeta signaling. Evaluation of the supernatants of the T cell hybridomas for cytokine production in response to the peptides revealed a close correlation between the induction of phosphorylation of TCR-zeta and the amount of cytokine induced. Selected analogue peptides were tested as tolerogens in neonatal mice. Analogues that did not induce the phosphorylation of zeta chain, such as B3 (CII 251-270s263F-->N), were completely unable to induce tolerance, while analogues that caused a partial phosphorylation, such as B6 (CII 251-270s267Q-->T) and A3 (CII 245-270s269P-->A), induced partial tolerance judged by intermediate degrees of suppression of arthritis. We conclude that discrete alterations in specific amino acid residues of antigenic peptides had profound effects on T cell signaling and that the signaling correlated with T cell cytokine secretion and T cell function in the induction of tolerance and suppression of arthritis.

Characterization of the T cell determinants in the induction of autoimmune arthritis by bovine alpha 1(II)-CB11 in H-2q mice.

Collagen-induced arthritis (CIA) is an experimental autoimmune disease elicited in genetically susceptible strains of mice by immunization with heterologous type II collagen. This experimental disease is mediated by the immune response of both T and B cells, and susceptibility is restricted by the class II molecules of the MHC. To study the T cell determinants of bovine type II collagen (CII) that mediate the autoimmune response in H-2q mice, we have identified a cyanogen bromide fragment of bovine CII, CII(124-402), that induces arthritis in DBA/1 mice. Using an overlapping set of peptides to map the T cell response to CII(124-402), we have determined that the I-Aq-restricted T cell response to this collagen fragment is mediated by a single immunodominant antigenic determinant. Consequently, this determinant plays a central role in promoting the production of the collagen-specific Abs and the induction of CIA in H-2q mice. Characterization of this immunodominant determinant revealed that the core residues required for T cell stimulation consists of only eight amino acids and is located at amino acids 260 through 267 of bovine CII. The systematic analysis of the contribution of each of these amino acids, in conjunction with sequences of other peptides known to bind to I-Aq, have allowed us to propose a peptide binding motif for the collagen arthritis susceptibility allele, I-Aq.

Genetic ablation of interferon-gamma up-regulates interleukin-1beta expression and enables the elicitation of collagen-induced arthritis in a nonsusceptible mouse strain.

OBJECTIVE: To determine whether the lack of interferon-gamma (IFNgamma) alters resistance to collagen-induced arthritis (CIA) in a nonsusceptible mouse strain, and if so, to identify changes in the antibody, cellular type II collagen (CII)-specific immune responses, and cytokine gene expression that might account for the altered susceptibility. METHODS: CIA-resistant C57BL/6 and C57BL/6 IFNgamma-/- mice were immunized with bovine CII in Freund's complete adjuvant (CFA) or in CFA alone. Animals were monitored for signs of arthritis for up to 80 days; arthritis severity was assessed visually and histologically. Sera were collected at various time points after immunization for measurement of anti-CII antibody levels. T cell responses to bovine CII were assessed in proliferation assays. Cytokine messenger RNA (mRNA) expression in lymph node cells and in synovial cells from arthritic paws was measured by RNase protection assays, and levels of cytokine protein production were determined by enzyme-linked immunosorbent assay. RESULTS: IFNgamma-/- mice developed a severe autoimmune arthritis that was dependent on immunization with CII. IFNgamma-/- mice produced significantly higher amounts of IgG1 and IgG2b antibody to the autoantigen, murine CII, compared with wild-type C57BL/6 mice and had an enhanced T cell proliferative response to bovine CII. Enhanced production of mature interleukin-1/beta (IL-1beta) protein was observed, but no significant changes in Th1 or Th2 cytokines. Although IL-6 and tumor necrosis factor alpha transcripts were clearly evident in the synovial cells from the arthritic paws of IFNgamma-/- mice, neither message was elevated to the levels measured for IL-1beta expression. Treatment of IFNgamma-/- mice with anti-IL-1beta significantly reduced the incidence and severity of the inflammation. CONCLUSION: Endogenous IFNgamma plays a role in the regulation of IL-1beta, in this model of autoimmune arthritis.

Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen.

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.