Abstract. Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium ([Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca"] i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system . The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 f 7 vs. 27 t 7, P < 0.01) . Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis . No calcium transients were detected in cells that bound but did not phagocytose beads . Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected : (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc B NDING and endocytosis of immune complexes by macrophages are mainly mediated by membrane receptors for the Fc portion of IgG (Fc receptors) (20) . Transmembrane signaling for the initiation of phagocytosis of bound immune complexes is not currently understood. Since calcium regulates the assembly and disassembly of cytoskeletal elements (7,18,22), it has long been proposed that a transient increase in the concentration of intracellular ionized calcium ([Ca2+]i) acts as a second messenger for phagocytosis . However, available studies with populations of macrophages have shown conflicting results concerning this possibility (8,14,26) . Studies of cell populations in which no calcium transients were observed may have been hampered by the fact that phagocytosis is an asynchronous event in different cells (14). Thus, calcium transients occurring in different cells at different times might have been averaged to undetectability as previously postulated by Kruskal and Maxfield (12). To overcome these difficulties, we used our previously described digital video imaging system (5,15,25) receptor-mediated phagocytosis (69.9 f 10.2 vs. 48.7 f 4 .7 s, P < 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 f 43 vs. 349 t 53 nM, P < 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis ; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the sign...

Human endogenous retroviruses (HERV) have emerged as a possible cause of systemic lupus erythematosus (SLE). We previously detected serum antibodies to the gng region of HERV clone 4-1 in patients with SLE, but not in normal volunteers. In the present study, we detected clone 4-1 messenger RNA (mRNA) in peripheral blood mononuclear cells (PBMC) from SLE patients and performed sequence analysis of the cDNA or genomic DNA from clone 4-1 in these patients. Clone 4-1 mRNA was detected in all of the SLE patients tested, although it was not found in normal controls. Sequence analysis of clone 4-1 in these SLE patients revealed inactivation of the stop codons in part of the gag region. In addition, a computer search of current sequence libraries revealed that the clone 4-1 gag genomic DNA from SLE patients was more highly homologous with the clone 4-1 sequence in chromosome 1 I from normal individuals when compared with the sequence of clone 4-1 integrated in the other chromosomes. It is possible that transcription of clone 4-1 from chromosome 1 1 occurs in SLE, and that the stop codon inactivation contributes to the translation of clone 4-1 gag proteins in patients with this disease.

Recent studies on epigenetics, including DNA methylation and its regulatory enzymes, seem likely to contribute to elucidation of the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE), although the relationship between DNA methylation and SLE has long been the subject of investigation. To obtain a deeper understanding of the role of DNA methylation in the induction of SLE, we reviewed the relationship between DNA methylation and SLE based on findings reported in the literature and our own data. Various studies, including ours, have indicated the possible importance of DNA methylation, especially hypomethylation, in the etiology of SLE. These epigenetic studies may give us clues towards elucidation of the pathogenesis of SLE and development of new therapeutic strategies for this disease.

The investigation of humanretroviruses has showndramatic progress since the discovery of human immunodeficiency virus (HIV). These studies have also contributed to the exploration of the role of retroviruses, including endogenous retroviruses, in the induction of autoimmune diseases such as systemic lupus erythematosus (SLE). This review describes the potential role of retroviruses in autoimmunity, based on recent findings including our own results. (Internal Medicine 40: 80-86, 2001)

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To investigate whether human endogenous retroviruses (HERV) contribute to autoimmune diseases, we prepared a recombinant p30gag protein derived from clone 4-1 of the HERV family, using a baculovirus-vector system. This p30gag protein (CA41B) was approximately 30 kDa, as expected, and reacted with antibodies for p30gag purified from both murine and feline leukemia virus. This result suggested that the antigenic determinant for p30gag was well conserved in CA41B. Analysis of serum antibodies to p30gag in patients with autoimmune diseases was done by Western blotting. CA41B detected anti-p30gag antibodies in 48.3% of systemic lupus erythematosus (SLE) patients, 35.0% of Sjögren's syndrome (SS) patients, and 33.3% of mixed connective tissue disease (MCTD) patients, whereas no anti-p30gag antibodies were found in healthy subjects. This suggested that HERV p30gag or other retroviral p30gag proteins possessing the same antigenic determinant as CA41B may play a role in these diseases. Although detection of antibodies to HERV p30gag in autoimmune diseases is indirect evidence that HERV proteins are involved, this study showed that patients with autoimmune diseases have antibodies to HERV p30gag using a recombinant HERV protein rather than synthetic peptides based on HERV or retroviral proteins of other species.

SUMMARYThe HIV-1 envelope glycoprotein (gp120) is known to induce antigen-specific and non-specific CD4 þ T cell anergy. We found that early T cell activation, as indicated by HLA-DP expression in the early G 1 (G 1A ) phase of the cell cycle, and the inhibition of mitogen-mediated IL-2 production induced by gp120, required TNF-a produced by gp120-stimulated macrophages. In the presence of an antibody to TNF-a, these changes induced by gp120 were inhibited, while recombinant TNF-a induced similar abnormalities of CD4 þ T cells, even in the absence of gp120. On the other hand, inhibition of the mixed lymphocyte reaction (MLR) in CD4 þ T cells by gp120, which may be related to gp120-mediated downregulation of CD4 expression on T cells and activation of protein tyrosine kinase p56 lck in CD4 þ T cells, was observed even in the absence of macrophage-derived TNF-a induced by gp120. These observations indicate that both TNF-a-dependent and independent events contribute to gp120-mediated CD4 þ T cell anergy, and TNF-a appears to play an important role in inducing CD4 þ T cell anergy in HIV-1 infection.

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