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News and Publications
Genetics of Autoimmunity and Gene Modifications in AIDS and Cancer
A.N. Theofilopoulos, D.H. Kono, D. Balomenos, S.R. Duncan, F. Kopti, H. Sabzevari, S. Soukchareun, U. Scheuring, S. Vidal
A major focus of this laboratory is the genetic basis of systemic autoimmunity in mouse models of spontaneous lupus erythematosus. We use congenic knockout mice to investigate the effects of specific genes on the disease process and microsatellite-based chromosomal maps to determine predisposing loci. We are also studying xenobiotic-induced systemic autoimmunity, gene modifications in HIV-infected cell lines, lung cancer biology, and other topics.
LOCI PREDISPOSING TO AUTOIMMUNITY IN FAS-DEFECTIVE MICE
Studies of Faslpr congenic mice established that the clinical characteristics and severity of disease depend on background genes; mice with lupus-prone backgrounds have more severe histologic manifestations than do mice with non--lupus-prone backgrounds. Thus, among the various strains of Faslpr mice, the most severe autoimmune disease develops in MRL-Faslpr mice. These mice have early onset of autoantibody production, glomerulonephritis, systemic vasculitis, arthritis, and 50% mortality at 5 months of age. In contrast, C57BL/6-Faslpr mice, which have the normal background, have much milder disease, characterized by less accumulation of lymphocytes, late onset of autoantibody production, and no or minimal histopathologic manifestations.
We did genome-wide searches to detect the additional loci required for severe lupus disease in Fas-defective mice. We found 4 loci with significant linkage to lymphadenopathy or splenomegaly on chromosomes 4, 5, 7, and 10: Lmb1, Lmb2, Lmb3, and Lmb4, respectively. Lmb1, Lmb 2, and Lmb3 were also linked to the production of antibodies to double-stranded DNA but not to glomerulonephritis; Lmb4 was associated with glomerulonephritis. Lmb2, Lmb3, and Lmb4 were inherited from the MRL background, but Lmb1 was derived from the B6-Faslpr. Nevertheless, each locus, regardless of the strain of origin, appeared to act in an additive manner, although certain combinations were more effective. Only a single suggestive locus on chromosome 1 could be correlated with arthritis. The identification of loci with highly significant linkage to disease manifestations in Faslpr strains make it possible to map and characterize new genetic defects that contribute to autoimmunity.
GENE KNOCKOUT LUPUS MICE
Analysis of T cells from 8-week-old unmanipulated MRL-lpr mice given bromodeoxyuridine in drinking water showed that higher proportions of CD4+ and CD8+ lymph node cells were dividing in MRL-lpr (15%) than in control MRL+/+ (3%) mice, and the proportion of cycling cells was even higher in the expanded double-negative subset of T cells (71%). Lymphadenopathy and other disease manifestations and the high rate of cycling for double-negative cells were severely reduced in mice deficient in p59fyn kinase. These findings indicate that the double-negative cells, contrary to previous claims, are functional, divide presumably in response to self-antigens, and contribute to the acceleration of disease in Fas-defective mice. Furthermore, the primary role of the p59fyn kinase appears to be signal transduction in double-negative cells that lack coreceptor molecules (CD4 and CD8).
We made 2 remarkable observations in MRL-lpr mice deficient in IFN- . First, hypergammaglobulinemia was maintained in homozygous IFN--/- mice with a switch from IgG2a to IgG1 predominance, but the dramatic decrease in levels of the dominant IgG2a autoantibodies to double-stranded DNA was not associated with compensatory increases in IgG subclasses associated with type 2 T helper cells. Second, early death and glomerulonephritis were prevented not only in IFN--/- mice, in which autoantibody levels and deposits of immune complexes in the kidney were severely reduced, but also in the heterozygous mice, even though autoantibody levels and deposits of immune complexes in the heterozygous IFN-+/- mice were equal to those in wild-type MRL-lpr mice. Expression of MHC class II molecules was severely reduced in both splenic mononuclear and renal epithelial cells in IFN--/- mice but only in the renal cells in IFN-+/- mice. These findings suggest that therapeutic interventions to reduce IFN- in lupus erythematosus may selectively affect pathogenic responses to double-stranded DNA without significantly compromising the ability to respond to exogenous antigens. They further suggest that even partial reduction of IFN- may curtail the deleterious effects of the cytokine in the afflicted organ after the initial deposition of immune complexes.
XENOBIOTIC-INDUCED LUPUS
Although genetic susceptibility is the major contributor to predisposition to autoimmune disease, environmental factors are also important determinants. In collaboration with K.M. Pollard, Department of Molecular and Experimental Medicine, and P. Hultman, Linkoping University, Sweden, we are defining the immunopathologic and genetic basis for systemic autoimmunity induced by the xenobiotic mercury. Exposure to mercury induces clinical features similar to those of spontaneous lupus erythematosus, including lymphoproliferation, hypergammaglobulinemia, and production of autoantibodies and immune complexes.
Current studies are directed to 2 main areas. First, genes relevant to the immunopathology of autoimmunity induced by mercury are being determined in knockout mice. Using mice that lack the gene for IL-4 or IFN-, we found that the prototypic mercury-induced autoimmunity mediated by type 2 T helper cells does not depend on IL-4 but on IFN-. Moreover, IFN- was required for antibody responses to both autoantigens and low doses of foreign antigen, presumably at the level of antigen presentation. This finding suggests that the resistance to mercury-induced autoimmunity in mice deficient in IFN- is due not to changes in the predominance of cellular vs humoral responses per se but to the requirement for IFN- to activate the immune system to respond to weak antigens. Studies are being done to define the molecular basis for the resistance of IFN---deficient animals to mercury-induced autoimmunity, particularly the roles of antigen-presenting cells, class II MHC molecules, and costimulatory molecules.
The second area of investigation is the genetic basis for the unique resistance of the DBA/2 strain to the systemic autoimmunity induced by mercury and other heavy metals. We have generated and phenotyped F(2) intercrosses between susceptible SJL/J and resistant DBA/2 strains. Initial mapping studies indicated 2 loci. One locus is located on chromosome 7 and overlaps 2 lupus-susceptibility loci derived from the MRL and NZW strains. Future studies on the derivation and analysis of congenic mouse lines, precise chromosomal mapping, and cloning and characterization of the resistance DBA/2 allele are planned.
PSORIATIC ARTHRITIS
Psoriasis is a chronic inflammatory skin disease often complicated by inflammatory arthritis. In collaboration with D. Boumpas, National Institutes of Health, we analyzed the gene repertoire for the variable region of the ß-chain for the T-cell antigen receptor among peripheral blood lymphocytes, skin, and synovium of patients with psoriatic arthritis. All patients had significant biases for the variable region of the ß-chain in the peripheral blood, and all had expansions common to both skin and synovium. Sequencing of complementarity-determining region 3 indicated that these expansions often consisted of oligoclonal or monoclonal populations. Although no ubiquitous nucleotide sequences were detected, 2 patients had identical sequences, and several highly homologous amino acid motifs were present in the skin and synovium among and between individual patients. These findings indicate an important role for cognate T-cell responses in the pathogenesis of psoriatic arthritis and suggest that the inciting antigen is identical or homologous between afflicted skin and synovium.
LUNG CANCER GENES
Tumor cells express a repertoire of genes different from those expressed by normal (nontransformed) tissues. Identification of these "malignant genes" may allow development of new diagnostic markers and provide insights into the pathogenesis of neoplasia. We have established a tissue bank of pairs of non--small cell lung tumors and syngeneic noncancerous lung from excised surgical specimens and bronchoalveolar lavage and thoracocentesis specimens from patients with a variety of malignant and nonmalignant diseases. To date, fragments of more than 20 genes have been tentatively identified and sequenced. Detailed analysis of differential expression of 4 distinct genes indicated 2 genes that are particularly interesting, and subsequent efforts have focused on further characterization of the structure and function of these genes.
EFFECTS OF FAS IN LUNG CANCER
We evaluated the function of Fas (CD95), an important stimulus for apoptosis, in human lung cancers. Surface expression of Fas was consistently upregulated during S and G2 or M phases and was affected by culture density in all but 1 of the 5 tumors. The extent of killing induced by cross-linking CD95 with monoclonal antibody was not related to expression of Fas but was highly influenced by the method of antibody treatment and prior incubation with IFN-. Fas stimulation preferentially induced apoptosis during S and G2 or M phases, and cell survivors were inhibited in their transit through S phase. Susceptibility to Fas-induced apoptosis was inversely related to mRNA levels of ICE and the ratio of BclXL to Bax and was directly proportional to expression of cyclin D3. Moreover, stimulation by antibodies to Fas caused significant, albeit highly varied, alterations in expression of other apoptosis genes, cyclins, and cyclin-dependent kinases. Pretreatment with antibodies to CD95 generally resulted in radiosensitization, although 1 lung cancer cell line paradoxically became radioresistant, a possible function of a unique Fas-stimulated depression of Bax levels.
These data show that Fas engagements have complex interactions in lung cancer cell dynamics and have substantive effects on transcriptional regulation in a large set of apoptosis and cell-cycle genes. Treating human lung cancers by manipulating the Fas pathway could ultimately be beneficial by inducing apoptosis or inhibiting cellular proliferation.
HOST GENE ALTERATIONS INDUCED BY HIV
In addition to constitutively expressed cellular proteins used by HIV type 1 (HIV-1), the virus presumably induces or suppresses expression of certain cellular genes. These transcriptional modifications may affect cellular proliferation, differentiation, transformation, and death and may contribute to the pathologic changes associated with HIV infection. In collaboration with D. Mosier, Department of Immunology, and J. Corbeil, University of California, San Diego, we are using differential RNA display analysis with a limited set of primers to characterize the effects of HIV infection on the expression of cellular genes.
Twelve differentially expressed bands, 6 upregulated and 6 downregulated, in CEM cells infected with HIV-1 were selected. Four of the 6 upregulated bands were HIV transcripts. RNase protection assays of the remaining 8 bands confirmed differential expression of 4 genes, including induction of a mariner transposase and moesin and suppression of  -nascent polypeptide-associated complex and mitochondrial heat-shock protein 75 in HIV-infected cell cultures. Furthermore, glioma pathogenesis-related protein was increased.
On the basis of this initial analysis, we estimated that expression of 3% of the host genes was altered by HIV-1 infection. Among the identified gene modifications, the induction of a mariner transposase may alter expression of cellular genes, whereas the enhanced expression of glioma pathogenesis-related protein suggests a role in the host cell response to viral infection. The increase in moesin may facilitate viral budding and uptake. Furthermore, the suppression of -nascent polypeptide-associated complex may promote translocation of HIV polypeptides into the endoplasmic reticulum, whereas the downregulation of mitochondrial heat-shock protein 75 may contribute to a cytopathic effect on mitochondria and possibly impair antigen presentation. These possibilities, as well as the role of viral genes in the expression of these cellular genes, are being analyzed.
PUBLICATIONS
Balomenos, D., Rumold, R., Stewart, T.A., Theofilopoulos, A.N. Interferon- is essential for lymphoaccumulation and lupus-like disease of MRL-lpr mice. J. Clin. Invest. 101:364, 1998.
Kono, D.H., Balomenos, D., Pearson, D.L., Park, M.S., Hildebrandt, B., Hultman, P., Pollard, K.M. The prototypic Th2 autoimmunity induced by mercury is dependent on IFN- and not Th1/Th2 imbalance. J. Immunol. 161:234, 1998.
Kuan, J., Kono, D.H. Tgfbr1 maps to chromosome 4. Mamm. Genome 9:95, 1998.
Lo, D., Aftahi, N., Reilly, C., Neal, H., Sim, B., Gascoigne, N.R.J., Kono, D.H., Wu, A., Schulman, S., Scott, B. Mapping genes regulating lymphocyte function: Correlations with autoimmunity? In: Current Directions in Autoimmunity, Vol. 1. Theofilopoulos, A.N. (Ed.). Karger, Basel, Switzerland, in press.
Norris, K., Norris, F., Kono, D.H., Vestergaard, H., Pedersen, O., Theofilopoulos, A.N., Moller, N.P.H. Expression of protein-tyrosine phosphatases in the major insulin target tissues. FEBS Lett. 415:243, 1997.
Scheuring, U.J., Corbeil, J., Mosier, D.E., Theofilopoulos, A.N. Early modifications of host cell gene expression induced by HIV-1. AIDS 12:563, 1998.
Shao, H., Kono, D.H., Chen, L.-Y., Rubin, E.M., Kaye, J. Induction of the Egr family of transcription factors during thymic selection. J. Exp. Med. 185:731, 1997.
Theofilopoulos, A.N., Kono, D.H. Mechanisms and genetics of autoimmunity. Ann. N.Y. Acad. Sci. 841:225, 1998.
Theofilopoulos, A.N., Kono, D.H. Murine lupus models: Gene-specific and genome-wide studies. In: Systemic Lupus Erythematosus, 3rd ed. Lahita, R.G. (Ed.). Academic Press, San Diego, in press.
Vidal, S., Kono, D.H., Theofilopoulos, A.N. Loci predisposing to autoimmunity in MRL-Faslpr and C57BL/6-Faslpr mice. J. Clin. Invest. 101:696, 1998.
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