Human alpha-fetoprotein contains potential heterodimerization motifs capable of interaction with nuclear receptors and transcription/growth factors.

Alpha-fetoprotein (AFP) serum levels in man have long been utilized as a tumor marker and as a birth defect screening agent in the clinical laboratory. Although the physiological role of AFP has remained obscure, the stereotypic carrier/transport function of a fetal counterpart to albumin has been attributed to this oncofetal protein. However, reports from a multitude of investigators in the last decade have provided a rationale to reconsider and extend the biological role of AFP to include cell growth modulation during development. Previously, a leucine zipper-like (heptad) motif, which mimicked that found in the steroid/thyroid receptor superfamily, was postulated for portions of the third domain of AFP. The present report proposes the presence of additional potential heterodimerization sites for the nuclear receptor superfamily members and other growth factors in the second and third domains of human AFP.

Signal transduction in Sjögren's syndrome T cells. Abnormalities associated with a newly described human A-type retrovirus.

OBJECTIVE: To study the effects of a novel A-type retrovirus, detected in cocultures of lip biopsy specimens from Sjögren's syndrome (SS) patients and a human T cell line, on the infected T cells. METHODS: Interleukin-2 (IL-2) and IL-6 secretion were measured by bioassay and enzyme-linked immunosorbent assay, respectively, in the infected and noninfected cell lines. Surface antigen expression was determined by flow cytometry, using monoclonal antibodies. Protein kinase C (PKC) activity was measured using an enzyme assay kit, and calcium mobilization was assessed with a fluorescent probe. RESULTS: Infected cells expressed less CD4 and IL-6 receptor, but more HLA-DR, compared with noninfected cells. Infected cells also produced less IL-2 and displayed reduced PKC activation and calcium mobilization. A similar defect in calcium mobilization was detected in T cells from SS patients. CONCLUSION: These data suggest a possible involvement of the newly described retrovirus in T cell abnormalities.

Detection of serum antibodies to retroviral proteins in patients with primary Sjögren's syndrome (autoimmune exocrinopathy).

Primary Sjögren's syndrome (SS) is considered a benign autoimmune disease; it is characterized by lymphoid infiltration of salivary and lacrimal glands, often accompanied by the presence of serum autoantibodies, particularly anti-Ro (SS-A) and anti-La (SS-B). There are important immunologic similarities between primary SS and acquired immunodeficiency syndrome. To investigate for a possible immune response to retroviral proteins in primary SS, we performed immunoblotting against human immunodeficiency virus-1 (HIV-1) proteins using sera from 47 patients with primary SS. Moderate-to-strong reactivity, suggesting the presence of serum antibodies, was found in 14 patients (30%). Of 120 normal subjects, only 1 showed moderate positivity. All 14 positive SS sera reacted against p24 (gag) but failed to react against gp41 or gp120 (env). This response did not reflect hypergammaglobulinemia since immunoglobulin concentrations among the 29 SS patients studied were the same in sera that contained and sera that did not contain anti-gag reactivity. Two sera also reacted against p17 gag. Four reacted against HIV-2 core proteins, but none reacted with core proteins of human T lymphotropic virus-I. Only 1 of the 14 sera reacted against Ro (SS-A), and 1 other reacted against La (SS-B). These results identify a subset of SS patients characterized by 1) the presence of serum antibodies to HIV-1 group-specific, but not type-specific, proteins, and 2) the relative absence of anti-Ro (SS-A) and anti-La (SS-B) autoantibodies. In this latter respect, these SS patients constitute a subpopulation that resembles patients with HIV-induced SS-like disease.

A conserved idiotype and antibodies to retroviral proteins in systemic lupus erythematosus.

22 of 61 systemic lupus erythematosus (SLE) patients produced antibodies to the p24 gag protein of HIV-1 demonstrated by Western blotting. 20 of these 22 patients (91%) also express the 4B4 idiotype (Id 4B4) previously identified on a human anti-Sm monoclonal antibody called 4B4. This represents an enrichment for this Id (seen in only 52% of SLE patients generally). Eight of these 22 SLE patients also have anti-Sm antibody activity. Sm partially inhibits the antibody binding of p24 gag suggesting immunologic cross-reactivity between the retroviral antigen p24 gag and the autoantigen Sm. Anti-Id 4B4 also inhibits p24 gag antibody binding by as much as 40%. Finally the monoclonal antibody 4B4 showed cross-reactivity to Sm and p24 gag. The following points emerge from our studies: (a) SLE patients make antibodies to p24 gag of HIV-1, (b) there is a relationship between immunity to p24 gag and a conserved idiotype, and (c) anti-Sm antibodies can cross-react with p24 gag.

Anti-peptide antibodies detect a lupus-related interspecies idiotype that maps to H chain CDR2.

Antibodies to the small nucleoprotein Sm occur spontaneously in human and murine systemic lupus erythematosus. Human and mouse monoclonal anti-Sm autoantibodies designated 4B4 and Y2 share an idiotype (Id) determinant located on the Ig H chain. To understand the molecular basis of this cross-reactivity, the VH regions of both antibodies were sequenced and analyzed for homology. The antibodies showed only 49.6% homology. The second complementary determining region (CDR2) was the most likely candidate for the Id site. To investigate this possibility, rabbit antiserum was made against a peptide corresponding to the CDR2 of 4B4. This antiserum was specific for the immunizing peptide and reacted weakly to a peptide corresponding to the CDR2 of Y2. Anti-CDR2 antibody bound to 4B4 and Y2 but not to other human and mouse mAb. Binding was directed at the H chain when analyzed by Western blots. Anti-CDR2 antibody blocked anti-Id antibody binding to 4B4 and Y2 by 58% and 24%, respectively. These studies suggest that this interspecies Id maps to the H chain CDR2 and that a conserved Id can occur within molecules that are otherwise radically different.

Characterization of the IL-2-receptor on rheumatoid arthritis synovial fluid T cells.

We studied the hypoproliferative response of synovial fluid (SF) T cells in rheumatoid arthritis (RA) using a mitogenic monoclonal antibody (MoAb) specific for the T-cell antigen receptor-associated CD3 complex. RASF T cells are defective in their proliferative response and in the induction of the Tac (p55) component of the IL-2-receptor (IL-2-R) when stimulated with anti-CD3 monoclonal antibody (MoAb). However, fresh RASF T cells bear demonstrable IL-2-R in cross-linking experiments which are not seen in unstimulated peripheral blood (PB). These receptors are functional since RASF T cells proliferate in response to recombinant IL-2 (rIL-2) better than fresh PB T cells from either normal or RA patients. Scatchard analysis indicates increased (4-fold) numbers of high affinity IL-2-R on (phytohaemagglutinin) PHA-activated RASF T cells as compared with comparably activated RAPB T cells. Phorbol myristate acetate (PMA) induces Tac antigen expression in RASF but does not lead to proliferation. The hyporesponsiveness of RASF T cells does not appear to result from lack of IL-2-R, lack of IL-2-R inducibility, or proliferative potential.

Autostimulatory growth factors produced by Sjögren's syndrome B cell lines.

Sjögren's syndrome (SS) is a systemic autoimmune disorder characterized by polyclonal hypergammaglobulinemia, autoantibody formation, and intense tissue infiltration by lymphoid and plasma cells. These patients have a predisposition to the development of monoclonal immunoglobulins and B cell lymphomas. To study the role of B cells in this disease, we established B cell lines from three patients with benign primary SS. These B cell lines grew spontaneously without any stimulation and expressed EBV nuclear antigen. The lines secreted an autostimulatory factor which had properties of a B cell growth factor. Peripheral blood B cells from SS patients, after culture for 3 days, secreted a similar factor which stimulated the proliferation of established SS B cell lines. These results suggest that circulating B cells in SS produce an autocrine growth factor that may contribute to lymphoproliferation and ultimately to the emergence of B cell lymphomas.

Autostimulatory factor produced by B-cell lines from patients with Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease with a predisposition to transform into a B-cell lymphoma. Stable B-cell lines were established (without exogenous stimulation other than fetal bovine serum) from the peripheral blood of three SS patients. These cell lines secreted immunoglobulin (either IgG, IgM or both) and expressed cytoplasmic immunoglobulin. They were positive for the B-cell markers Leu 12 and Leu 16, and also for HLA-DR and the transferrin receptor. The cells lacked CD3 and the IL-2 receptor. Supernatants from these cell lines had autostimulatory activity. When 24-h culture supernatants were added to freshly cultured peripheral blood mononuclear cells, the proliferation index was 2-3 times higher as compared to cells cultured with HB101 medium alone. This autostimulatory activity can be attributed to a B-cell growth factor since these supernatants were also able (1) to support the growth of BD9 cells and (2) to augment the proliferation of PB B cells preactivated with S. aureus Cowan strain I. Furthermore, the supernatants did not contain IL-1, IL-2, or gamma-interferon. Thus, B cells that grow spontaneously from the peripheral blood of SS patients spontaneously produce a B-cell growth factor. This factor could contribute importantly to the autoantibody production, tissue lymphoid infiltration and B-cell lymphoma seen in this disease.

Estrogen induces normal murine CD5+ B cells to produce autoantibodies.

Females have better humoral immune responses and are more susceptible to autoimmune diseases than males. Normal female mice (C57BL/6J, C3H/HeJ, and NZW) have significantly increased spontaneous autoimmune plaque-forming cells (APFC) to mouse erythrocytes pretreated with bromelain (Br-ME) in spleen, peritoneal exudate cell, and bone marrow compared to their male counterparts. A minor subpopulation of B cells, CD5+ B, is thought to produce this autoantibody. As determined by dual color flow cytometry, increased APFC to Br-ME in females is not due to quantitative increase of CD5+ B cells. Rather, it is due to increased numbers or percentages) of CD5+ B cells producing these autoantibodies, because CD5+ B cells from females produced greater numbers of APFC to Br-ME than equal numbers of cells derived from males. The increased autoantibody production in females can be attributed to the effect of estrogen on the immune response because this hormone markedly augments APFC to Br-ME in intact or orchidectomized males. Male hormone had little effect. Importantly, estrogen did not increase the numbers of B or CD5+ B cells but augmented the ability of B cells to produce this response. This was verified when a T cell-depleted B cell fraction or fluorescence-activated cell sorter purified CD5+ B cells from estrogen-treated mice proved more efficient in the production of APFC to Br-ME. These results suggest that the number of CD5+ B cells committed to produce autoantibodies to Br-ME is increased under the influence of estrogen. This is the first demonstration that estrogen can augment the production of natural autoantibodies in normal mice. The overall augmented humoral immune responses in females and the B cell hyperactivity in female predominant autoimmune diseases appears to be due to estrogen.

The expression and function of CD3 and CD5 in patients with primary Sjögren's syndrome.

We studied the expression and function of 2 cell surface markers that induce T cell activation, CD3 and CD5, in 19 patients with primary Sjögren's syndrome (SS). The expression of CD3+ lymphocytes was normal, but CD3 function was moderately reduced, as measured by anti-CD3-induced T cell proliferation. Anti-CD3-induced stimulation of T cell help for Ig production and non-major histocompatibility complex-restricted (natural) killing were normal. In contrast, there was reduced expression and function of the CD5 molecule on peripheral blood lymphocytes of the SS patients. The ratio of CD5+ to CD3+ lymphocytes was 0.45 in SS patients compared with 0.85 in normal subjects, indicating that the CD3+ cells are relatively CD5-deficient in SS patients. Anti-CD5 monoclonal antibody did not augment suboptimal anti-CD3 stimulation of whole peripheral blood lymphocytes or of purified T lymphocytes from SS patients, which indicated impaired functioning of the CD5 molecule. A significant impairment in proliferation was found in response to phorbol myristate acetate and to ionomycin in combination, suggesting defective intracellular signaling. Findings from serial studies of individual patients suggested that normal or low expression of CD5 on T cells can be stable over periods as long as 2 years. However, in 2 patients who required systemic therapy, a correction of the CD5 lymphocyte abnormality occurred in association with clinical remission, which suggests the potential reversibility of this condition. Our findings suggest a possible defect in intracellular signaling in primary SS, related in part to the CD5 molecule, which may provide a molecular explanation for the T cell hyporesponsiveness that is characteristic of this disease.

Defective induction of T-cell help and natural killing following anti-CD3 stimulation of autoimmune lymphocytes.

Anti-CD3 monoclonal antibody (MoAb) stimulates T cells in normal peripheral blood to proliferate and develop cytotoxic activity against NK-sensitive tumor cell lines. We now find that anti-CD3 MoAb also generates cytotoxic activity against a cell line (MEL-21) resistant to classical NK cell killing. After activation in vitro with anti-CD3 MoAb for 18 h, normal peripheral blood mononuclear cells (PBMNC) develop more HLA-DR-positive helper than suppressor T cells, manifest a functional helper effect as measured by increased IgG synthesis (P less than 0.01), as well as kill MEL-21 target cells. PBMNC from rheumatoid arthritis (RA) patients respond normally but mononuclear cells from rheumatoid arthritis synovial fluid (RASF) respond poorly. PBMNC from systemic lupus erythematosus (SLE) patients also respond poorly to anti-CD3 stimulation. Thus, the ability of anti-CD3 to stimulate IgG production and generate enhanced natural cytolytic activity are defective in both RASF and SLE lymphocytes.

Abnormalities of T cell activation in the rheumatoid synovium detected with monoclonal antibodies to CD3.

The chronic inflammation of rheumatoid arthritis (RA) is associated with hypofunction of synovial fluid (SF) T cells. We studied the mechanisms leading to this abnormality using a mitogenic monoclonal antibody specific for the T cell receptor-associated CD3 complex. We found that SF cells are defective in their response to anti-CD3 antibodies as measured by proliferation, generation of natural cytotoxicity, and induction of the Tac (p55) component of the IL2 receptor. Nevertheless, these cells do bear functional IL2 receptors and are more responsive to IL2 than are resting peripheral blood T cells. In searching for a mechanism to explain the reduced IL2 production, we found that polyamines (whose oxidation products can down-regulate proliferation and IL2 production) are elevated in RA cells from both blood and SF. We postulate that the chronic activation of RA T cells triggers this feedback loop which constitutes a defensive mechanism aimed at reducing the T cell driven autoimmune and inflammatory process.

A shared B-T cell phenotype in autoimmune mice bearing the lpr gene.

MRL-lpr mice and MRL-+/+ mice are identical except for the presence of an autosomal recessive lymphoproliferation gene (designated lpr) in the former. Mice bearing the lpr gene develop autoimmune and lymphoproliferative abnormalities. An antigenic marker designated 14D10, characteristically expressed on the surface of Lyt-2+ T cells and B cells of normal mice, is expressed in unusually high levels on Thy-1+ cells of lpr mice which are Lyt-2-. In younger lpr animals, 14D10+ cells are a minor subpopulation of Ia+ cells which, when expanded in diseased animals, continue to express Ia. 14D10+ cells from lpr mice are elevated in fetal spleen and adult bone marrow (BM) but are absent on pre-B cells in the BM. Medullary thymocytes of lpr mice are enriched in 14D10+ cells compared to congenic (+/+) controls. Although 14D10 appears to be present on the activated, proliferating T-cell population, coculture of lpr cells with IL-2 leads to minimal proliferation. 14D10+ Lyt-2- T cells can be isolated from normal spleens, indicating the lpr gene may be responsible for the disregulated proliferation of a minor cell subset. The functional significance of this molecular complex is still undetermined.

Altered natural killer and natural cytotoxic cellular activities in lpr mice.

Three strains of mice bearing the autosomal recessive lpr gene (MRL, C57BL/6, and C3H) that had spontaneously developed a lupus-like disease were studied sequentially for functional natural killer (NK) and natural cytotoxic (NC) cell activity. Natural killing was impaired in spleen and bone marrow cells from all the lpr strains, as well as from the congenic strain MRL--+/+, which develops a late onset lupus-like disease. The NK cell activity was found to be depleted as early as 2 months of age in all lpr strains, and decreased further with age. NK activity was augmentable by Poly I:C and interleukin 2 (IL-2), suggesting that the residual cells can respond to NK modulators. In contrast with NK cell activity, NC activity was not decreased in lpr mice but could be augmented by IL-3-rich supernatants. The spontaneous decrease in NK cell activity was associated with an increased autologous plaque-forming cell (APFC) response to bromelin-treated mouse red blood cells, which is produced primarily by B cells possessing the Ly-1 phenotype (Lyt-1+ B). When NK cell activity was increased by exogenous administration of Poly I:C, the APFC response diminished. Treatment of spleen cells with anti-asialo GM1 prior to Poly I:C treatment resulted in a decreased NK response but increased both APFC and Lyt-1+ B cells. The possible regulation of autoreactivity by NK cells is discussed.

Immunotherapy for myasthenia gravis: a murine model.

In vivo therapy with monoclonal antibody (mAb) GK1.5, which recognizes a glycoprotein antigen designated L3T4 on murine helper T lymphocytes, either prevented or suppressed the development of murine lupus, autoimmune encephalomyelitis, and collagen arthritis. The L3T4 antigen in the mouse is analogous to the human Leu-3/T4 antigen expressed on helper T lymphocytes, because they both participate in the T cell response to class II major histocompatibility complex (MHC) antigens. Class II MHC genes and I-A antigens mediate murine experimental autoimmune myasthenia gravis (EAMG) induced by acetylcholine receptor (AChR) autoimmunity. We studied the efficacy of mAb GK1.5 as an immunotherapeutic agent for murine EAMG. Therapy with mAb GK1.5 not only suppressed established autoimmunity to AChR but also prevented loss of muscle AChR in mice with EAMG. Moreover, permanent remission of clinical muscle weakness was induced if mAb GK1.5 therapy was initiated after the onset of clinical disease. Because the function of the Leu-3/T4 determinant on human helper T lymphocytes is analogous to the murine L3T4 determinant, use of antibody to the Leu-3/T4 determinant as an immunotherapeutic agent may provide a way to control the progression of human MG.

Phorbol ester induction of Ia and a shared B-cell/T-cell phenotype in normal but not autoimmune (lpr) mice.

Tumour-promoting phorbol diesters are mitogenic for lymphocytes and induce differentiation of B and T cell lines as well as promyelocytic leukaemia cells. This paper demonstrates that 12-O-tetradecanoyl-13-acetate (PMA), when cocultured with normal murine bone marrow cells (BMC), significantly augments an antigenic cell-surface determinant called 14D10. This antigen is present constitutively in the majority of bone-marrow lymphocytes of autoimmune lpr mice. PMA has little enhancing effect when cocultured with lpr BMC. In addition, Ia antigenic determinants are increased by PMA in normal but not lpr BMC. Retinoic acid (RA) and PMA act synergistically both to increase 14D10 and to enhance the stimulatory ability of target lymphocytes as measured by proliferation in an autologous mixed lymphocyte reaction (AMLR). We suggest that lpr mice have persistent expression of gene products like 14D10 that are usually repressed in normal adult mice. These gene products can be activated in normal mouse bone marrow by PMA which acts through a Ca2+-dependent phospholipid-dependent C protein kinase. The in vivo enhanced expression of 14D10 in lpr mice suggests activation by some mechanism or factors yet to be described.

Effects of short-term administration of sex hormones on normal and autoimmune mice.

The effects of short-term administration (2 to 4 wk) of sex hormones on the immune system of normal (C57BL/6) and autoimmune (C57BL/6-lpr, C3H/lpr, B/W) strains of mice were investigated. Both estrogen (E2) and testosterone (Te) had significant effects on the numbers of T and B cells as well as on the density of cell surface antigens as demonstrated by flow cytometry. For example, Te depleted Thy-1.2+ thymocytes in normal mice and brought about a shift to lower density cells. Lyt-2+ cells appeared to be the main target cells of hormonal modulation in normal and autoimmune mice. Both sex hormones significantly depleted these cells in the thymus but had differential effects in the peripheral lymphoid organs, particularly in the spleen. In general, E2 depleted Lyt-2+ cells, whereas Te increased or maintained this subpopulation of cells in spleen and lymph nodes. Similarly, the suppressor cell activity and IL 2 production on a per cell basis in E2-treated animals was diminished, whereas Te-treated animals had normal or enhanced activity. The relevance of these findings to differential sex susceptibility in autoimmune diseases is discussed.

Increased Ia expression, T lymphocyte subset abnormalities and autoimmunity in murine strains bearing the lpr gene.

Defects in cellular communication are fundamental to the development of autoimmune disease. Modulation of immunoregulatory events can be mediated by cellular expression of Ia antigens. We have analysed, by flow cytometry, the Ia antigenic levels on cells from mice expressing the lpr gene and their congenic counterparts. Surface Ia expression is dramatically increased on bone marrow, thymus, lymph node and spleen cells from lpr mice even prior to characteristic lymph node and spleen enlargement. In addition, IL-2 production abnormalities occur in the low density Lyt 1 subset of Thy 1.2 positive cells of normal mice which may be the counterpart of the majority cell type of lpr lymphocytes. Treatment of lpr mice with low dose whole body irradiation (300 rad) decreases lymphadenopathy, autoantibodies, proteinuria and the resident Ia positive cell population while increasing survival. We conclude that lymphoid alterations induced by irradiation reflect a recovery of immunological control associated with suppression of autoimmune manifestations.

Suppression of lymphoproliferation and auto-immunity by elimination of a radiosensitive bone marrow cell in mice bearing the lpr gene.

The autosomal recessive lpr gene (lymphoproliferative) has been bred into several normal strains of mice. Although the time of disease onset may vary, all of these lpr mice develop hypergammaglobulinaemia, antibodies to nucleoproteins, and massive lymphoproliferation. The abnormalities do not appear in their normal congenic counterparts, which lack the lpr gene. We studied the effects of sublethal whole-body X-irradiation (300 rads) on disease features and lymphocyte subpopulations by using flow cytometry. H-2k strains, C3H and MRL/++ and their autoimmune lpr counterparts, were killed at 1, 2, 4, 8 and 24 weeks after irradiation, which was given at 6-8 weeks of age. In the lpr mice, lymphoproliferation, autoimmunity, mortality and number of Ia+ cells were greatly reduced in the irradiated mice compared with nonirradiated controls. Abnormal cytofluorometric patterns seen with lpr thymocytes were corrected by the low-dose irradiation treatment. In addition, lethally irradiated lpr mice reconstituted with syngeneic bone marrow from low-dose irradiated mice exhibited retarded development of autoimmune disease. We conclude that the lymphoid alterations induced by X-irradiation reflect a recovery of immunologic control associated with suppression of autoimmune manifestations.