Role of the major histocompatibility complex class II Ea gene in lupus susceptibility in mice.

The gene(s) encoded within major histocompatibility complex (MHC) act as one of the major genetic elements contributing to the susceptibility of murine systemic lupus erythematosus (SLE). We have recently demonstrated that lupus susceptibility is more closely linked to the I-E- H-2(b) haplotype than to the I-E+ H-2(d) haplotype in lupus-prone BXSB and (NZB x BXSB)F1 hybrid mice. To investigate whether the reduced susceptibility to SLE in H-2(d) mice is related to the expression of the MHC class II Ea gene (absent in H-2(b) mice), we determined the possible role of the Ea gene as a lupus protective gene in mice. Our results showed that (i) the development of SLE was almost completely prevented in BXSB (H-2(b)) mice expressing two copies of the Ead transgene at the homozygous level as well as in BXSB H-2(k) (I-E+) congenic mice as for H-2(d) BXSB mice, and (ii) the expression of two functional Ea (transgenic and endogenous) genes in either H-2(d/b) (NZB x BXSB)F1 or H-2(k/b) (MRL x BXSB)F1 mice provided protection from SLE at levels comparable to those conferred by the H-2(d/d) or H-2(k/k) haplotype. In addition, the level of the Ea gene-mediated protection appeared to be dependent on the genetic susceptibility to SLE in individual lupus-prone mice. Our results indicate that the reduced susceptibility associated with the I-E+ H-2(d) and H-2(k) haplotypes (versus the I-E- H-2(b) haplotype) is largely, if not all, contributed by the apparent autoimmune suppressive effect of the Ea gene, independently of the expression of the I-A or other MHC-linked genes.

In vivo immunosuppression by targeting a novel protease receptor.

Membrane receptors for blood proteases govern the clotting and fibrinolytic cascades, regulate signal transduction and control the growth of mesenchymal cells. Despite their importance in the development of vascular injury, it is unclear whether these mechanisms participate in the generation of an immune response. Here we report that targeting a factor Xa receptor, designated effector cell protease receptor-1 (EPR-1), with antisense oligonucleotide or with a monoclonal antibody (mAB 2E1) inhibited CD3/T-cell receptor-dependent lymphocyte proliferation. Immunosuppression was mediated by abolishing cytokine production and down-modulating membrane expression of the interleukin (IL)-2 receptor. In vivo administration of mAb 2E1 to severe-combined-immunodeficient mice injected with human peripheral blood leukocytes suppressed production of human immunoglobulin, abolished graft-versus-host disease, and protected these xenochimaeric mice from Epstein-Barr-virus-induced human lymphoproliferative disease. These observations indicate a new role for protease receptors in the regulation of the immune response, and identify a potential target for therapeutic immunosuppression in humans.

Characterization of hu-PBL-SCID mice with high human immunoglobulin serum levels and graft-versus-host disease.

A chimeric model consisting of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL) has recently been described (bu-PBL-SCID mice). These reports indicated a limited reconstruction of the transferred human immune system and functionality of the human graft. Herein we described modifications of the PBL transfer method that minimize transfer time and cellular manipulations, leading to a more effective population of SCID mouse recipients. Severe combined immune deficiency mice given 15 x 10(6) PBL had human IgG serum levels reaching 2 to 5 g/l, and all mice had detectable human anti-tetanus toxoid antibody levels when they received cells from donors with such levels. These transfers were associated also with clinical and histologic evidence of graft-versus-host disease, suggesting responsiveness of the human graft in the recipients. When Epstein-Barr virus seropositive (EBV+) donors were used, the chimeric mice also showed a high incidence of fatal lymphoproliferative disease 1 to 3 months after transfer of 15 x 10(6) PBL. The high level of immunoglobulin synthesis and immunoresponsiveness of the human cells with this transfer procedure may expand the use of these chimeric mice for the manipulations of human immune cells in vivo.

The hu-PBL-SCID mouse model. Long-term human serologic evolution associated with the xenogeneic transfer of human peripheral blood leukocytes into SCID mice.

We present a 2-year serologic analysis of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL, hu-PBL-SCID mice). After 10-20 x 10(6) PBL transfer, human IgG serum levels generally increased in the SCID mouse recipient for 2 months, and thereafter decreased without returning to zero for at least 2 years. Great variability existed between different hu-PBL-SCID mice with regard to Ig serum levels even when derived from the same donor's PBL aliquot. The ratio of IgM to IgG serum levels was lower in hu-PBL-SCID mice than in the donors. The half-life of human IgG in the SCID mouse is shorter than in the human (8 days vs 23 days), suggesting a much higher production of IgG than expected from serum levels. The majority of hu-PBL-SCID mouse sera analyzed by high resolution electrophoresis had a smear appearance suggestive of diverse human Ig, generally with superimposed multiple faint mIg. Few mice developed strong human mIg, associated with lymphoproliferative diseases. In the hu-PBL-SCID mouse model, the transfer of cells from donors making antibody with defined specificity against TT and nuclear antigen resulted in the appearance of these antibodies in only a minority of the recipients.

Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries.

Antibodies are usually prepared from recently boosted animals and reflect ongoing immune responses. In humans, this is restrictive as ethical constraints generally prevent antigen-boosting. Therefore the rich memory compartment of human antibody responses remains largely untapped. Severe combined immune deficiency (SCID) mice populated with human cells allow the stimulation of human antibody memory without the usual constraints. Here we show how peripheral blood lymphocytes can be stimulated by antigen to produce large secondary responses after transfer to SCID mice. Specific monoclonal human Fab fragments can then be isolated from the mice by repertoire cloning even when the human donor's last contact with antigen was more than 17 years ago.

An autosomal recessive gene that delays expression of lupus in BXSB mice.

We report the generation and serologic, cellular, histologic, and genetic characteristics of a BXSB/MpJScr substrain, termed BXSB/MpJScr-ll/ll, that has lost early-life male lupus disease. Classic genetic analysis suggested that delayed disease expression results from the action of a single autosomal recessive gene. This putative gene, referred to as ll (long-lived), causes a significant delay in expression of autoimmune serology (total serum IgG and anti-nuclear antibodies levels), monocytosis, and of immune complex-mediated histopathologic changes such as glomerulonephritis, arteritis, and myocardial infarction. Presumably as a consequence of the delayed immunopathology male BXSB/MpJScr-ll/ll mice live three to four times longer than regular BXSB/MpJScr. This strain might be useful for analysis of single genes responsible for severe autoimmune disease expression.

Transfer of human systemic lupus erythematosus in severe combined immunodeficient (SCID) mice.

To study the role of peripheral blood leukocytes (PBL) in the pathogenesis of human systemic lupus erythematosus (SLE), we transferred PBL from 5 SLE patients into 15 severe combined immunodeficiency (SCID) mice. Such reconstituted mice showed long-term presence of auto-antibodies characteristic of the donor in their sera, as well as human immunoglobulin deposition, and in some cases mouse C3, in the renal glomeruli. SCID mice repopulated with PBLs from normal donors do not develop serologic abnormalities or immunodeposits. It is concluded that human SLE serology and some associated renal changes can be reproduced solely by PBL transferred from afflicted patients, and that SCID-human-SLE mice may serve as an in vivo laboratory model for the study of human SLE.

Correlations of autoimmunity with H-2 and T cell receptor beta chain genotypes in (NZB X NZW) F2 mice.

Accelerated autoimmunity as expressed by the classical autoimmune strain mouse (NZB x NZW)F1 is thought to be the result of major histocompatibility complex (MHC)-associated NZW genes acting on a genetic predisposition for autoimmunity as expressed by the NZB mouse. To evaluate more accurately both H-2 and T cell receptor (TcR) beta chain involvement in F1 disease, we studied the segregation of NZB (H-2d, TcRB) and NZW (H-2z, TcRW) haplotypes of these genetic elements and the development of autoimmunity in (NZB x NZW)F2 generation mice. F2 mice with the H-2d/z genotype lived shorter average life-spans and expressed elevated levels of antibodies to gp70, ssDNA and dsDNA, while those with the TcRW/W genotype (homozgous for the NZW TcR deletion) expressed elevated levels of autoantibodies but had relatively long life-spans. On the other hand, mice with the TcRB/B genotype (homozygous for the NZB TcR) produced consistently low levels of autoantibodies but died at an early age. The most severely affected F2 population were the mice carrying both the TcRB/B and H-2d/z alleles. These mice died on an average within the first 5 months of life, but produced the lowest levels of antibodies to gp70, single-stranded DNA and double-stranded DNA. These data confirm the contribution of NZW H-2-linked genes to accelerated autoimmunity in the F1 hybrid and, furthermore, define NZB TcR-linked components as primary developers of this phenomenon. They also suggest a limited, if any, contribution of both the NZW TcR deletion and traditional autoantibodies to F1 accelerated autoimmunity.

Association of elevated serum glycoprotein gp70 with increased gp70 immune complex formation and accelerated lupus nephritis in autoimmune male BXSB mice.

BXSB male mice, which spontaneously develop a systemic lupus erythematosus (SLE) like disease, were the only strain to have a significant incidence of abnormally elevated levels of gp70 in sera. Concentrations of gp70 in some mice were more than 10 times (greater than 500 micrograms/ml) those of young BXSB and any other murine strain. The presence of high serum levels of gp70 was significantly associated with hepatic sinusoidal lymphocytosis, a high incidence of which was only observed in male BXSB mice. Serum levels of gp70-anti-gp70 immune complexes were greatly increased in mice with high levels of gp70, presumably associated with increased anti-gp70 antibody production. Such mice developed fatal glomerulonephritis significantly earlier than those with lower levels of gp70. These results suggest that (1) hepatic inflammation of unknown aetiology occurring uniquely in male BXSB mice during the course of their SLE may be responsible for the enhanced expression of serum gp70 antigen, because of its nature as an acute phase reactant and (2) enhanced expression of gp70 antigen is associated with increased formation of anti-gp70 antibodies and exacerbation of lupus nephritis in male BXSB mice.

Analysis of vascular lesions in murine SLE. I. Association with serologic abnormalities.

In murine SLE, two different vascular lesions can develop. A necrotizing polyarteritis (NPA), exclusively found in MRL/I mice, is characterized by a dense infiltration of PMN and fibrinoid necrosis of the arterial wall. The second, a degenerative vascular lesion, occurs in a low incidence in all SLE mice, except the (NZW X BXSB)F1 (WBF1) male, in which its incidence is 100%. This lesion shows subendothelial deposits of immunoglobulins with minimal or no inflammatory or proliferative reaction. This degenerative vascular disease (DVD) is predominantly localized in the coronary arteries and is highly correlated with myocardial infarction. Serologic analysis revealed that NPA in MRL/I mice was associated with relatively late development of high levels of autoantibodies and circulating immune complexes; DVD in WBF1 mice was associated with an early onset of autoantibody production of a low magnitude that gave rise to a persistent low level of circulating immune complexes. Characterization of circulating immune complexes in MRL/I mice showed these complexes were mainly of intermediate size (7S-19S) and contained predominantly anti-DNA antibodies. In WBF1 mice, complexes were barely detectable and contained mostly anti-gp70 antibodies. Elution of kidneys showed that the major antibody deposited in MRL/I mice has an anti-DNA specificity, whereas in WBF1 animals, the major antibody was anti-gp70. Furthermore, a 10 times greater amount of immunoglobulins could be eluted from WBF1 hearts with DVD than from MRL/I and BXSB hearts. Additionally, we found that the lack of an inflammatory reaction in DVD was not because of a preferential deposition of noncomplement-fixing IgG1 antibodies nor could it be related to a defective inflammatory response, because WBF1 mice had an undiminished reverse passive Arthus reaction throughout their lives. It is concluded that NPA develops secondary to high levels of autoantibodies with a concomitant rise in immune complexes, whereas DVD is associated with sustained low levels of circulating immune complexes.

Low-calorie diet selectively reduces expression of retroviral envelope glycoprotein gp70 in sera of NZB x NZW F1 hybrid mice.

The effect of dietary restriction on the expression of retroviral envelope glycoprotein, gp70, and the formation of gp70-anti gp70 immune complexes was investigated in lupus-prone NZB x NZW F1 hybrid mice. Restricting total calorie intake from the usual 20 to only 10 calories per day after weaning markedly reduced serum levels of both free and antibody-complexed gp70, prevented renal disease, and increased the life spans of these mice. The reduction in serum gp70 was evident after only 2 wk of feeding these animals the low-calorie diet, and the concentration remained virtually unchanged throughout the course of 10 mon experimentation. However, serum concentrations of the major structural protein, p30, of endogenous retroviruses were not altered by restricting calories. Amounts of the serum glycoprotein, haptoglobin, decreased parallel to those of gp70 but amounts of albumin did not. These results suggest that the expression of gp70 in serum is controlled independently of the production of complete viral particles, and regulated by a mechanism similar to that for other serum glycoproteins, such as haptoglobin.

Retroviral gp70 immune complexes in NZB x NZW F2 mice with murine lupus nephritis.

NZB x NZW (NZB x W) F1 hybrid mice spontaneously develop a disease most prominently characterized by immune complex glomerulonephritis (GN), which seems to be associated with both antibodies to DNA and to the serum retroviral envelope glycoprotein, gp70. To evaluate the contribution of each of these autoimmune responses to the pathogenesis of the GN, we studied NZB x W F2 mice in which the two responses appeared to segregate relatively independently. Use of this model permitted analysis of possible correlations between each response and the G.N. The presence of circulating anti-gp 70-complexed gp70 correlated significantly with the development of fatal GN and one could predict the course of renal disease by computing the rising serum levels of gp70 complexed with antibodies. In contrast, the presence of free antibodies to either double-stranded or single-stranded DNA was not significantly associated with the development of fatal GN. This association of anti-gp70 antibody production with these animals' early death from GN strongly suggests that the gene(s) governing production of antibodies to serum retroviral gp70 may be one of the major genes responsible for spontaneous renal disease segregated in NZB x W F2 generations.

Induction of high serum levels of retroviral env gene products (gp70) in mice by bacterial lipopolysaccharide.

In the present study, mice each given a single intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) responded with increased serum levels of the major envelope glycoprotein, gp70, of endogenous retrovirus. Concentrations of gp70 in their sera began to increase 4 hr after LPS injection, reached maximal 5- to 15-fold increases after 12--24 hr, and returned to the preinjection levels within 3 days. This response occurred only in the strains characterized by high base line levels of serum gp70 (greater than 10 micrograms/ml) such as NZB, NZB X NZW F1, BXSB, MRL, NZW, DBA/2, LG, 129(GIX+), and C57BL/6(GIX+). However, strains such as DBA/1, C3H/St, BALB/c, C57BL/6(GIX-), and 129(GIX-) with lower base line levels of serum gp70 (less than 5 micrograms/ml) made little or no response. This serum gp70 induced by LPS was structurally similar to the gp70 of NZB xenotropic virus that is dominantly expressed in sera from virtually all strains of mice. However, (i) the induced gp70 was virion-free; (ii) xenotropic virus was not isolatable from BXSB, MRL/1, or 129(GIX+) mice injected with LPS; and (iii) amounts of the major structural viral protein, p30, did not increase correspondingly in sera. All of these findings indicate that the increased expression of serum xenotropic viral gp70 in response to LPS did not result from activation of replication-competent xenotropic virus. In addition, the serum gp70 response to LPS was abolished by simultaneous inoculation of an inhibitor of protein synthesis, D-galactosamine. These results strongly suggest that LPS selectively stimulates synthesis of the env gene product, gp70, of NZB xenotropic virus but other viral gene products.

Identification of retroviral gp70 and anti-gp70 antibodies involved in circulating immune complexes in NZB X NZW mice.

Retroviral gp70 and anti-gp70 antibodies were isolated from circulating immune complexes (IC) of 7-10-mo-old (NZB X NZW)F1 mice, after which the nature and origin of this gp70 (IC-gp70) and the immunologic characteristics of these anti-gp70 antibodies (IC-anti-gp70) were investigated. Immunochemical and structural analyses of IC-gp70 demonstrated that among multiple immunologically related gp70 expressed in all mice, the IC-gp70 had characteristics similar to those of NZB xenotropic viral gp70 (NZB-X1 gp70) that is commonly present in sera of virtually all strains of mice. The study of binding by IC-anti-gp70 antibodies to retroviral gp70 from various sources showed that the IC-anti-gp70 were primarily directed to NZB-X1 gp70 as well as serum gp70. These data strongly suggest that the abnormality of murine strains with systemic lupus erythematosus causing them to produce antibodies to their own xenotropic viral gp70 and to form IC with serum gp70 is not based on their expression of an unusual type of gp70, but rather their ability to make an antibody to NZB-X1 gp70, probably as a result of their immunologic dysfunction.

Selective suppression of retroviral gp70-anti-gp70 immune complex formation by prostaglandin E1 in murine systemic lupus erythematosus.

The effect of pharmacologic quantities of prostaglandin E1 (PGE) was investigated in three strains of mice (NZB X NZW, MRL/1, and BXSB) that spontaneously develop lupus-like glomerulonephritis. PGE-treatment prolonged survival and retarded the glomerular deposition of immune complex (IC) and the development of glomerulonephritis in NZB X NZW and MRL/1 mice, but did not similarly protect BXSB mice. Changes in the responsive strains correlated well with reduced amounts of circulating gp70 complexed with anti-gp70 antibodies compared with untreated controls, although total concentrations of gp70 (free and complexed) detectable in sera were similar in both groups of mice. The results strongly suggest that: (a) PGE selectively suppressed the immune response to retroviral gp70, (b) PGe had little effect on the quantity or quality of anti-DNA antibodies but did reduce the deposition of anti-DNA containing IC in the kidneys, and (c) gp70 IC appear to play an important role in the pathogenesis of glomerulonephritis in murine systemic lupus erythematosus.

Male determined accelerated autoimmune disease in BXSB mice: transfer by bone marrow and spleen cells.

Autoimmune disease in BXSB mice progresses more rapidly in male animals. We have investigated the cellular basis of this effect by transferring male and female bone marrow and spleen cells into male and female lethally irradiated BXSB recipients. The rate of development of disease was measured by the overall mortality rate, mortality from glomerulonephritis, and development of serologic abnormalities. We found that the pace of disease in the BXSB sex chimeras was determined entirely by the sex of the donor of the transferred cells. Lethally irradiated BXSB animals receiving male cells had rapidly progressive disease, whether the recipients were themselves male or female, whereas female-cell recipients of either sex had slowly progressive disease. The male-specific effect that accelerates autoimmune disease in the BXSB is thus not hormonally mediated, but rather is expressed in the hematopoietic stem cell populations.

Association of circulating retroviral gp70-anti-gp70 immune complexes with murine systemic lupus erythematosus.

Endogenous retroviral gp70 was investigated as a participant in the pathogenesis of a lupus-like disease that spontaneously develops in four kinds of mice (NZB, NZB x W MRL/1, and male BXSB). Sera from these strains contain a heavy form of gp 70 that varies in sedimentation rates from 9S to 19S in sucrose density gradient analysis and appears with the onset of disease and persists throughout its course. Immunologically normal strains of mice do not develop rapidly sedimenting gp70 by 8-10 mo of life. The fact that the heavy gp70 is selectively absorbed with anti-IgG antibodies or with Staphylococcus aureus protein A suggests that it is complexed with antibodies. The incidence and quantities of these gp70 ICs rise with the progression of disease in all strains with lupus. These findings suggest that Ig-complexed heavy gp70 may be involved in the pathogenesis of glomerulonephritis of mice with SLE.

Increased spontaneous polyclonal activation of B lymphocytes in mice with spontaneous autoimmune disease.

Early in life, mice of four kinds [NZB, (NZB X NZW)F1, MRL/1, and male BXSB] with autoimmune disease spontaneously produced far more (greater than 3 S.D.) anti-hapten antibody-forming cells in spleens and greater concentrations of anti-hapten antibodies in sera than immunologically normal strains of mice (AKR, BALB/c, C57BL/6, DBA/1-J, DBA/2J, LG/J, 129, NZW, and female BXSB). This increased nonspecific antibody production by the abnormal animals' B cells correlated well with the spontaneous development of anti-single-stranded DNA antibodies, but not with serum levels of the viral envelope glycoprotein, gp70. These results suggest that the spontaneous formation of autoantibodies in mice whose immunologic disorder is manifested by a lupus-like disease may result from polyclonal activation of B cells by endogenous or exogenous B cell activators.