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Cytokines play a critical role in regulating the quantitative and qualitative characteristics of cellular and humoral immune responses. As such, these molecules would be expected to inhibit or promote the initiation and perpetuation of autoimmune diseases, including lupus disease.

Previous studies with blockers or stimulators of IFN- suggested that this major cytokine plays an important role in the pathogenesis of lupus disease. To conclusively assess the role of IFN-, we created congenic MRL-lpr mice homozygous and heterozygous for the deletion of the gene for this cytokine. Early death was prevented and glomerulonephritis was severely reduced in both types of mice compared with control animals. In mice homozygous for the deletion, hyperglobulinemia was maintained, but with a switch from IgG2a to IgG1 predominance, and the predominantly IgG2a autoantibodies to double-stranded DNA were severely reduced without concomitant increases in other IgG isotypes. Despite the reduction in glomerulonephritis in mice heterozygous for the deletion (50% reduction in IFN- levels compared with the levels in controls), the levels of autoantibody were unaffected, indicating the importance of additional locally exerted disease-promoting effects of IFN-. Mice homozygous for the deletion also had reduced lymphadenopathy associated with decreases in the dominant double-negative (CD4^-CD8^-) T-cell population. Moreover, macrophages (antigen-presenting cells) from the homozygous mice expressed markedly lower levels of MHC class I and II molecules than did control mice.

These findings indicate that hyperproduction of IFN- is required for the development of lupus disease. The high levels of this cytokine presumably increase MHC expression and autoantigen presentation to otherwise quiescent, nontolerant, low-affinity, anti-self T cells and promote local inflammatory processes. These results suggest that even partial reduction of IFN- may inhibit or reduce immunopathologic changes associated with lupus disease.

ROLE OF P59^FYN IN DOUBLE-NEGATIVE T-CELL ACTIVATION AND EXPANSION

The severe spleen and lymph node hyperplasia of MRL-lpr lupus mice that have a defect in the Fas gene (apoptosis gene) is characterized by expansion of double-negative (CD4^-CD8^-) T cells. These T cells appear to be derived from CD8⁺ precursors and are thought to be functionally inert. The latter conclusion, however, is not supported by the activation phenotype of double-negative cells.

To further characterize the functional status of the lpr double-negative T cells, we performed in vivo labeling experiments in wild-type MRL-+/+ and mutant MRL-lpr as well as p59^fyn-/- MRL-lpr mice. Fyn transduces T-cell receptor--derived signals by association with the CD3 zeta-chain, whereas the other major proximal tyrosine kinase, p56^lck, transduces signals by association with CD4 or CD8. Therefore, the only kinase available for activation of double-negative cells is Fyn.

Analysis of T cells from 8-week-old mice given a DNA precursor in drinking water for 9 days showed that higher proportions of CD4⁺ and CD8⁺ lymph node cells were dividing in lpr (15%) than in +/+ mice (3%), and the proportion of cycling cells was even higher in the double-negative (71%) and CD4⁺B220⁺ (54%) lpr subsets. The CD4⁺B220⁺ cells also expand in lpr mice, but to a much smaller degree than do the double-negative cells. Radionuclide chase experiments showed that activation and division in most double-negative cells were initiated subsequent to the cells' precursor CD8⁺ stage. Lymphadenopathy and other manifestations of disease were greatly reduced in Fyn^-/- lpr mice concomitant with decreased proportions of double-negative cells (from 77% to 20%). Division of double-negative cells was also severely reduced in Fyn^-/- lpr mice, whereas all other subsets were unaffected. A similar phenomenon was observed in double-negative liver cells of Fyn^-/- mice of normal background.

We conclude the following: (1) Double-negative lpr T cells, rather than being end-stage, retain the capacity to be activated and repeatedly divide before reaching an anergic or replicative senescence stage. (2) The Fyn kinase associated with the CD3 zeta-chain is important for double-negative cell signal transduction. (3) Double-negative cells are highly dependent on Fas participation for apoptosis. (4) Double-negative cells contribute to the early development of the serologic and histologic features of the MRL-lpr lupus disease.

CYCLIN KINASE INHIBITORS AND REPLICATIVE SENESCENCE

A general characteristic of lupus-prone mice (and humans) is the expedited accumulation of large numbers of presumably self-reactive T cells with the phenotypes of activated and memory cells. The mechanisms by which these cells escape apoptosis have not been elucidated. We used CD4⁺CD44^hi cells, which have the phenotypes of activated and memory cells, obtained from male BXSB mice with early-life severe lupuslike disease to investigate the status of the cell cycle and susceptibility to apoptosis and to determine the role of corresponding genes in the survival of these cells.

Most of the rapidly accumulating memory phenotype CD4⁺CD44^hi cells of 4-month-old male BXSB mice were arrested in G₁ of the cell cycle. With advanced age, the CD4⁺CD44^hi cells switched from predominantly cycling to predominantly noncycling. Moreover, the CD4⁺CD44^hi cells of older mice were refractory to proliferation and apoptosis induced by antibodies to CD3. These cells also expressed high levels of certain cyclin-dependent kinase inhibitors.

We propose that in autoimmunity, self-reactive T cells are subjected to successive rounds of activation and division that eventually lead to a buildup in cyclin-dependent kinase inhibitors. High levels of these inhibitors cause refractoriness to further activation, impaired entry into the cell cycle, and resistance to apoptosis, a situation akin to replicative senescence. These findings are important not only in systemic autoimmunity but also in many other situations in which the immune system is subjected to repetitive immune responses against foreign antigens or self-antigens, as in AIDS, aging, rheumatoid arthritis, and other conditions.

QUANTITATIVE TRAIT LOCI

Previous studies in our laboratory determined several autoimmune susceptibility loci for the (NZB X NZW)F₁ hybrid, one of the major animal models of systemic lupus erythematosus, which also include the MRL-Fas^lpr and BXSB strains. We have now identified non-Fas^lpr susceptibility loci in mice homozygous for the apoptosis-defective Fas^lpr mutation by using MRL-Fas^lpr (severe autoimmune disease) and C57BL/6-Fas^lpr (B6-Fas^lpr; minimal late-onset disease) mice. A panel of 274 (MRL-Fas^lpr x B6-Fas^lpr)F₂ intercross mice was used to study pathologic features associated with systemic lupus erythematosus or inflammatory arthritis. We found significant differences between the two parental backgrounds in lymphadenopathy, splenomegaly, antibodies to double-stranded DNA, glomerulonephritis, and arthritis.

Using interval mapping, we found that four quantitative trait loci, designated Lmb1, Lmb2, Lmb3, and Lmb4, on chromosomes 4, 5, 7, and 10, respectively, were linked with lymphadenopathy and/or splenomegaly. Lmb1, Lmb2, and Lmb3 were also linked to the production of antibodies to double-stranded DNA but not to glomerulonephritis; Lmb4 was associated with glomerulonephritis. Several other loci that met the criteria of suggestive significance were also detected, including a single locus associated with arthritis on chromosome 1 and two others associated with glomerulonephritis on chromosomes 6 and 19. Lmb2, Lmb3, and Lmb4 were inherited from the MRL background, but interestingly, Lmb1 was derived from the B6 background. Each locus acted in an additive manner regardless of its strain of origin, although certain combinations were more effective. Thus, the mild disease in the B6 background may be attributable to the presence of only a single major susceptibility locus.

These findings clearly show that genetic heterogeneity for lupus susceptibility genes may sometimes include genes from so-called normal strains, provide an explanation for why crosses of the same strain to different backgrounds may yield different sets of susceptibility loci, and suggest that analysis of F₂ intercrosses may be preferable for autoimmune susceptibility studies because genetic contributions from both parental strains can be defined. The exact nature of genes corresponding to these susceptibility loci must await the development of congenic animals and further refinements of the chromosomal locations of the loci. The determination of quantitative trait loci with highly significant linkage to disease manifestations in Fas-^lpr strains will make it possible to map and clone new genetic defects that contribute to autoimmunity.

PUBLICATIONS

Baccala, R., Gonzalez-Quintial, R., Kono, D.H., Theofilopoulos, A.N. Multiprobe RNase protection assay and sequence enrichment nuclease assay for TCR analysis. In: The Human Antigen T Cell Receptor: Selected Protocols and Applications. Oksenberg, J. (Ed.). R.G. Landes, Georgetown, TX, 1997, p. 86.

Balomenos, D., Rumold, R., Theofilopoulos, A.N. The proliferative in vivo activities of lpr double-negative T cells and the primary role of p59^fyn in their activation and expansion. J. Immunol. 159:2265, 1997.

Beijleveld, L.J.J., Damoiseaux, J.G.M.C., Roglic, M., Theofilopoulos, A.N., van Breda Vriesman, P.J.C. Effector cells in cyclosporin-A-Induced autoimmunity: Both CD4 and CD8 T cells generate autoimmune disease upon adoptive transfer. Transplantation 62:1468, 1996.

Chowers, Y., Marsh, M.N., deGrandpre, L., Nyberg, A., Theofilopoulos, A.N., Kagnoff, M.F. Increased proinflammatory cytokine expression in the colonic mucosa of coeliac disease patients in the early period after gluten challenge. Clin. Exp. Immunol. 107:141, 1997.

Duncan, S.R., Elias, D.J., Roglic, M., Pekny, K.W., Theofilopoulos, A.N. T-cell receptor biases and clonal proliferations in blood and pleural effusions of patients with lung cancer. Hum. Immunol. 53:39, 1997.

Hui, S., Kono, D.H., Chen, L.-Y., Rubin, E.M., Kaye, J. Induction of the early growth response (Egr) family of transcription factors during thymic selection. J. Exp. Med. 185:731, 1997.

Kono, D.H., Owens, D.G., Wechsler, A.R. Jak3 maps to chromosome 8. Mamm. Genome 7:476, 1996.

Kono, D.H., Theofilopoulos, A.N. Genetics of murine SLE. In: Dubois' Lupus Erythematosus, 5th ed. Wallace, D.J., Hahn, B.H. (Eds.). Williams & Wilkins, Baltimore, 1996, p. 119.

Roglic, M., MacPhee, R.D., Duncan, S.R., Sattler, F.R., Theofilopoulos, A.N. T cell receptor BV gene repertoires and clonal expansions of CD4 cells in patients with HIV infections. Clin. Exp. Immunol. 107:21, 1997.

Sabzevari, H., Propp, S., Kono, D.H., Theofilopoulos, A.N. G₁ arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated memory phenotype CD4⁺ cells of lupus mice. Eur. J. Immunol. 27:1901, 1997.

Theofilopoulos, A.N. Mechanisms and genetics of autoimmunity. In: The Role of Immune Mechanisms in Cardiovascular Disease. Schultheiss, H.-P., Schwimmbeck, P.L. (Eds.). Springer-Verlag, New York, 1997, p. 3.

Theofilopoulos, A.N. Systemic lupus erythematosus: Experimental models. In: Encyclopedia of Immunology, 2nd ed. Delves, P.J., Roitt, I.M. (Eds.). Saunders, Philadelphia, in press.

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