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B-cell hyperactivity is the general and cardinal feature of murine and human systemic lupus erythematosus (SLE), the prototype organ-non-specific autoimmune disease. The defect(s) responsible could be intrinsic to B cells, secondary to T-cell or macrophage abnormalities resulting in excessive help or deficient suppression, or could stem f rom qualitative/quantitative abnormalities of autoantigens. Here Argyrios Theofilopoulos and his colleagues review the immunopathological and cellular abnormalities observed in murine lupus and the factors currently thought to play a role in normal B-cell proliferation, differentiation and Ig class switching. Next month, in a second article, they discuss abnormalities in B-cell response to and/or overproduction of T-cell-derived accessory signals which may explain the generalized B-cell hyperactivity associated with mouse and human lupus.

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.

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 × 106 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.

ANTIBODIES are usually prepared from recently boosted animals and reflect ongoing immune responses1–6. In humans, this is restrictive as ethical constraints generally prevent antigen-boosting. Therefore the rich memory compartment of human antibody responses remains largely untapped7. Severe combined immune deficiency (SCID) mice8 populated with human cells9–11allow 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.

HE IMMUNOPATHOLOGIC study of kid­ ney disease, glomerulonephritis, and more recently expanded to tubulointerstitial nephritis, has closely followed the technological advances in biomedicine and been periodically punctuated by perceptive observations and amazingly accurate interpretation and postulation. The role of immu­ nologic events in the pathogenesis of glomeru­ lonephritis was recognized both in the experimen­ tal laboratory and in the clinic in the first few years of this century. In 1900, Lindemann induced al­ buminuria and uremia in rabbits by injection of guinea pig anti-rabbit nephrotoxic serum. 1 A num­ ber of immunologic studies in the next two dec­ ades indicated first that the responsible nephro­ toxic antigen was in the renal cortex and later more specifically in the glomeruli. 2 In the follow­ ing two decades, a number of the most eminent immunologists such as Masugi, Smadel, and Kay analyzed the pathologic and immunologic aspects of this disease, concluding that morphologically the renal changes were quite similar to some cases of human glomerulonephritis, that the disease oc­ curred in distinct acute and chronic stages, and finally that the host's immune response to the het­ erologous nephrotoxic serum was the crucial ele­ ment in the autologous chronic progressive phase of the disease. 3-5 These findings afforded us as complete an immunopathologic picture of this dis­ ease model as pre-World War II technology would permit. During this same 40-year period, a very dif­ ferent trail of immunopathologic investigation de­ veloped that also implicated immunologic events in glomerulonephritis. This line of study began not in the experimental laboratory, but in the clinic where von Pirquet made some unbelievably astute

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.

Interstitial nephritis in rats immunized with heterologous tubular basement membrane. Interstitial nephritis developed in Brown Norway (BN) and Lewis/BN (L/BN) F 1 hybrid rats within two weeks after immunization with bovine cortical tubular basement membrane (TBM) in complete Freund's adjuvant (CFA) or pertussis adjuvant (PA). The histologic lesion began as a polymorphonuclear leukocyte infiltrate and quickly progressed to a primarily mononuclear cell infiltrate with tubular atrophy and tubular epithelial cell degeneration, interstitial and periglomerular fibrosis, and only minimal glomerular abnormalities. It was most severe in BN rats immunized with bovine TBM in PA. Direct immunofluorescence of kidneys revealed linear deposits of IgG along TBM of all proximal tubules and 10 to 20% of distal tubules, and occasionally along the Bowman's capsule. Indirect immunofluorescence revealed anti-TBM antibody in serum and renal eluates. There were no anti-GBM antibodies. Only minor abnormalities of renal function were detected. None of ten additional rat strains similarly immunized developed nephritis. Two strains, F344 and August, did not produce antibody in response to immunization; three strains, Lewis, Wistar-Furth (W/F) and Maxx, developed antibodies but did not possess an antigen on their TBM cross-reactive with the immunizing bovine TBM; and five strains, ACI, Buffalo, Wistar, W/F (fz) and DA, had only small quantities of anti-TBM antibody deposited in their kidneys and did not develop nephritis. Nephrite interstitielle chez des rats immunises avec de la membrane basale tubulaire heterologue. Une nephrite interstitielle se developpe chez les rats Brown Norway (BN) et les hybrides Lewis/BN (L/BN) F 1 en deux semaines apres l'immunisation avec de la membrane basale tubulaire corticale bovine (TBM) dans de l'adjuvant de Freund complet (CFA) ou de l'adjuvant de pertussis (PA). Les lesions histologiques commencent par une infiltration de polynucleaires et deviennent rapidement un infiltrat de cellules mononuclees associe a une atrophie tubulaire avec une degenerescence des cellules epitheliales tubulaires, une fibrose interstitielle et periglomerulaire et des anomalies glomerulaires minimes. Ces images sont plus severes chez les rats BN immunises avec la TBM bovine dans le PA. L'immunofluorescence directe des reins montre des depots lineaires d'IgG le long des TBM de tous les tubes proximaux et de 10 a 20% des tubes distaux et parfois le long de la capsule de Bowman. L'immunofluorescence indirect revele des anticorps anti-TBM dans le serum et les eluats de rein. Il n'y a pas d'anticorps anti-GBM. Seules des anomalies mineures de la fonction renale sont observees. Aucune des dix autres souches de rats immunises n'a developpe de nephrite. Deux souches, F344 et August, n'ont pas produit d'anticorps en reponse a l'immunisation; trois souches, Lewis, Wistar-Furth (W/F) et Maxx, ont produit des anticorps mais n'avaient pas d'antigenes sur leurs TBM aui reagissent avec la TBM bovine immunisante; cinq souches, ACI, Buffalo, Wistar, W/F (fz) et DA, avaient seulement de petites quantites d'anticorps anti TBM deposees sur leurs reins ent n'ont pas developpe de nephrite.

MRL/l mice spontaneously develop an arthritis very similar in many respects to human rheumatoid arthritis. A detailed morphologic and serologic analysis of this disease revealed the following: (a) a 75% incidence of synovial and periarticular inflammation, very similar to human rheumatoid arthritis, in 5-6 mo-old females, (b) close associations between presence of joint inflammation and subsynovial and/or periarticular vasculitis, and (c) a close correlation between presence of circulating IgM rheumatoid factor (RF) and demonstrable synovial and/or joint pathology, i.e., 95% of mice with significant levels of IgMRF had synovitis and/or arthritis.

In an attempt to determine whether genes involved in T cell antigen recognition are structurally abnormal and thereby promote murine systemic lupus, we analyzed the structural integrity of the D, J, and C region elements of the T cell receptor alpha and beta chain genes in all major lupus strains and several normal strains. Within the limits of restriction fragment length polymorphism analysis, all strains had an identical genomic organization, except the NZW mice, in which a deletion of the C beta 1-D beta 2-J beta 2 elements was found. Sequence analysis of NZW genomic elements containing this deletion placed its probable origin within the first exon of C beta 1, and extending to a complementary region within the first exon of C beta 2. The significance of this abnormality in the pathogenesis of systemic autoimmune disease remains to be determined.

The several kinds of mice that spontaneously develop acute systemic lupus erythematosus (SLE)—BXSB males, MRL/I males and females, and (NZB x W)F1 females—have a 15–20% incidence of degenerative vascular disease (DVD) and myocardial infarcts (MI) in which the affected coronaries contain deposits of immunoreactants, presumably in the form of immune complexes. Among the F1 hybrid crosses of SLE mice, only the (NZW x BXSB)F1, (W x B)F1 male has a significantly higher incidence of DVD/MI (80%). Search for possible causes of this high incidence of myocardial infarcts revealed several unique features of this mouse: hypertension, thrombocytosis, and early onset of circulating immune complexes and glomerulonephritis. Our attempts to prevent this DVD/MI focused on: reduction of hypertension, prevention of thrombosis, and immunosuppression. Immunosuppression by Cytoxan resulted in almost complete prevention of both the SLE disease and DVD/MI. Administration of bretylium, an antihy-pertensive and anti-arrhythmic agent, resulted in reduction of blood pressure and the severities of glomerulonephritis, DVD, and MI; phritis, DVD, and MI; it also slightly reduced the levels of circulating immune complexes and leukocytosis. Of the 4 antithrombotic agents used, only aspirin showed some reduction in the incidence of DVD/MI and delay of glomerulonephritis-associated mortality.

To define the polymorphism and extent of the mouse immunoglobulin kappa (Igk) gene complex, we have analyzed restriction-enzyme digested genomic DNA from 33 inbred strains of mice with labeled DNA probes corresponding to 16 Vk protein groups (1 of them previously undescribed) and the Jk/Ck region (V, variable; J, joining; C, constant). These probes detected between 1 and 25 distinct restriction enzyme fragments (REF) that appeared in up to eight polymorphic patterns, thus defining eight mouse Igk haplotypes. The investigated portion of the Vk repertoire was estimated to encompass between 60 and 120 discernable Vk gene-containing REFs. In contrast to mouse VH gene families, several Vk gene families defined by these probes appeared to overlap. This observation has implications for Vk gene analyses by nucleic acid hybridization and raises the possibility that the Vk gene complex is a continuum of related sequences.

Three IgM monoclonal anti-DNA antibodies were produced by hybridoma techniques from an MRL - lpr lpr mouse using denatured DNA (dDNA) as the selection antigen. All three antibodies also bound poly(dT), poly(rA), and the single-stranded random copolymer poly(dI,dT), and each antibody displayed a unique preference for a limited array of other ribo- and deoxyribopolynucleotides based on direct binding as well as inhibition studies. Inability to identify a common primary structure in the polynucleotides reactive with each antibody suggested that higher ordered structures may be important. This notion was supported by the finding that oligomers of thymidine of 25–30 nucleotides or less were ineffective in blocking antibody binding to dDNA or poly(dT). However, deliberate destabilization of putative secondary structures by decreasing counterion concentration and increasing temperature had little effect on antibody binding to poly(dT). Since the antigenic polynucleotides in general contain little known secondary structure and considerable flexibility, antibody binding may be accompanied by local conformational changes in the polynucleotide that result in a better fit to the antibody combining site.

The effects of five distinct bacterial lipopolysaccharides (LPS) on the induction of polyclonal IgM and IgG antibodies, including polyclonal autoantibody formation, were investigated in several strains of mice. Injections of most LPS preparations that contained polysaccharide transiently induced only IgM polyclonal antibodies. However, LPS from Salmonella minnesota R595 (R595 LPS), which had a particularly high content of lipid A but lacked O-antigen polysaccharide, induced a markedly prolonged IgM and IgG polyclonal antibody response in mice, including athymic nude mice, but not in LPS-unresponsive C3H/HeJ mice. Polyclonal IgM and IgG production peaked in sera on day 8 and day 15, respectively, and remained higher than control values 2 mo after the injection. The IgG induced by R595 LPS was strictly restricted to IgG2b and Igg3 subclasses in normal mice. In contrast, in athymic nude mice which have normally lower levels of IgG1 and IgG2a than normal mice, R595 LPS stimulated the production of all the IgG subclasses and reconstituted serum levels of IgG1 and IgG2a up to, but not higher than, control values of normal mice. These findings suggest that different mechanisms regulate production of each IgG subclass after stimulation with LPS.

Antigen-antibody complexes are being increasingly implicated in kidney diseases, collagen disorders, a variety of viral, bacterial, and parasitic infections, and malignancies. Perhaps their most im...

The flavivirus dengue and the arenavirus Junin are both associated with a hemorrhagic shock syndrome in man. We have demonstrated the replication of these viruses in vitro in both rabbit and human endothelial cells by viral titers and immunofluorescent antibody studies. Rabbit endothelium established in continuous culture was derived from vena cava, while human cells in primary culture were derived from umbilical veins. In rabbit endothelium, dengue-2 virus passaged through monkey kidney monolayer cells (LLC-MK2) or human lymphoblastoid cells (raji) produced significantly more virus than the seed obtained from suckling mouse brain (MB). Inoculation of actively dividing, subconfluent human endothelial cells with the LLC-MK2 degue virus produced higher viral titers than inoculation of confluent cells. The appearance of Junin virus was delayed beyond that of dengue virus in rabbit endothelial cells although equivalent titers of virus were produced. In human cells, Junin virus was less productive than dengue virus and produced characteristic cycles of virus release. This is the first direct evidence for replication of human hemorrhagic fever viruses in endothelial cells.

The mRNA encoding heavy and light chains of a hybridoma-derived monoclonal IgM, kappa anti-immunoglobulin (rheumatoid factor) and an IgG3, kappa anti-histone autoantibody from systemic lupus erythematosus and arthritis-prone MRL/Mp-lpr/lpr mice have been molecularly cloned, and the nucleotide sequences corresponding to their variable regions have been determined. To investigate whether autoantibodies with specificities frequently observed in lupus disease might share common structural components, the sequences obtained in this study have been compared with those of a monoclonal MRL/Mp-lpr/lpr IgM, kappa anti-DNA autoantibody previously analyzed in our laboratory (J. Exp. Med. 1985. 161: 805). The 3 immunoglobulins employed different heavy chain variable region (VH) genes belonging to the large J588 VH gene family, kappa light chain variable region (V kappa) genes from 3 different V kappa groups, and different diversity and joining segments. Our findings suggest that murine lupus-associated autoantibodies of different specificities do not have genetic components in common to signal their self-reactive nature and are encoded by a large number of immunoglobulin gene elements.

A comprehensive analysis of the immunocompetency of three murine strains displaying spontaneous autoimmune disease was conducted at various times during the lifespan of these mice. Qualitative and quantitative comparisons have been made for antibody responses to well defined hapten-carrier conjugates between young and old autoimmune as referenced against nonautoimmune inbred mice of the same ages. Responses of intact autoimmune and normal murine strains in two immunoglobulin classes, i.e., IgG and IgE, failed to reveal any general pattern of abnormal immune reactivity that could be ascribed solely to the autoimmune phenotype; grossly deficient antibody-producing capacities that were observed in old mice of the MRL/1 strain do not necessarily relate to the existence of autoreactivity in such animals. In addition to studies in intact mice, the immunocompetency of isolated B lymphocyte and regulatory T lymphocyte populations of these autoimmune mice were assayed for functional abnormalities by utilizing adoptive transfer systems. No significant differences were observed between young and old autoimmune mice and normal control strains either in the antibody-producing abilities of adoptively primed hapten-specific B lymphocytes or in regulatory functions of antigen-specific helper and suppressor T cells. Even in those circumstances in which intact mice showed marked deficiencies in antibody responses, i.e., old MRL/1, lymphocytes removed from the native environment of such mice displayed normal reactivity patterns in adoptive transfer situations. Of special note are the results presented herein that clearly demonstrate that antigen-specific regulatory T cell functions of both helper and suppressor types are, in fact, normal in both old as well as young animals of all three autoimmune murine strains. These findings raise serious questions about previous concepts concerning the existence of any single defect or combination of immunoregulatory defects as universal pathogenetic mechanisms in the development of autoimmune diseases.

Autoimmune disease appears to be a consequence of the generation of self-reactive antibodies. The relationship between these autoantibodies and antibodies directed against exogenous antigens has fostered much recent work, especially on the murine models of systemic lupus erythematosus and rheumatoid arthritis, as Reinhard Kofler and his colleagues review here. While the complexities surrounding the origin of self-specific antibodies are still to be completely unravelled, it appears that lupus autoantibody expression may not result from defects in lg germline genes nor in mechanisms generating antibody repertoires (variable region gene selection, rearrangement, somatic mutation) but follows the same general principles governing responses to foreign antigens.

B lymphocyte hyperactivity in systemic lupus erythematosus (SLE)-prone mice, originall considered a result of imbalances in T-cell subsets, more recently has been attributed to intrinsic abnormalities of B cells. In our experiments cited here, we used a variety of systems to assess the surface phenotypic and functional characteristics of B cells from several SLE strains (NZB, NZB/W, BXSB, MRL/Mp-lpr/lpr). Generation of Ig isotype diversity follows normal pathways in all these SLE strains. B cells from newborn BXSB and MRL/Mp-lpr/lpr mice, as in immunologically normal mice, do not reexpress surface Ig (sIg) after modulation with anti-Ig. In contrast, B cells of newborn New Zealand mice do reexpress sIg after anti-Ig-induced modulation. The rates at which sIg-anti-Ig complexes cap and become endocytosed in all SLE strains are within normal limits. B cells from SLE strains are stimulated mitogenically by F(ab') 2 anti-μ and lipopolysaccharide with indices no different from those of normal B cells. The ontogenic development of Ia + and Lyb5 + cells is normal with the frequency of positive cells and density of alloantigens normal or slightly elevated, respectively. SLE and normal strains are much alike in expression of retroviral envelope gp70 antigen on surfaces of lymphocytes. Anti-gp70 antibodies fail to stimulate mitosis in cells of either SLE or normal strains. However, the frequency of B-cell colony-forming splenocytes is several fold higher in autoimmune mice compared to controls. Expression of acceptor sites for helper messages and of acceptor sites for suppressor messages on B cells is within normal limits in all SLE mice as revealed by the effects of immune response enhancing anti-Lyb3 serum and immune response suppressing anti-Lyb7 serum, respectively. As a whole, these results reaffirm the generalized hyperactivity and advanced maturation of B cells of SLE mice but do not reveal any common surface or functional characteristics which might be responsible for the B-cell abnormality.

B cell hyperactivity, hypergammaglobulinemia, and autoantibody expression, the hallmarks of systemic lupus erythematosus, might be associated with structural abnormalities within the Ig heavy chain variable region (Igh-V) gene complex. The Igh-V loci from several lupus-prone mouse strains, their ancestors, and other nonautoimmune mice were therefore analyzed by restriction fragment length polymorphisms with DNA probes corresponding to seven VH gene families. These seven families comprise the majority of the known polymorphic murine VH gene repertoire, including some involved in autoantibody generation. Our study showed that the Igh-V loci from lupus and haplotype-matched nonlupus mice resulted in essentially identical restriction fragment patterns, a finding which suggests that the Igh-V gene complex does not carry a primary defect responsible for autoimmune disease.

MRL 1 mice develop an autoimmune, lupus-like disorder characterized by massive proliferation of T cells and rapidly fatal immune complex nephritis. Prostaglandin E1 (PGE1), in pharmacologic quantities, retarded the development of this syndrome. The beneficial effects included: (1) increased lifespan, (2) prevention of peripheral T lymphoid hyperplasia, (3) preservation of T-cell mitogenic responses, (4) inhibition of immune complex-mediated glomerulonephritis, and (5) reduction in the amounts of circulating gp70-anti-gp70 immune complexes and of IgG1 and IgG2b. These studies suggest that the protective effects of PGE1 are related to the inhibition of lymphoid hyperplasia and the consequent prevention of renal disease.

An important problem in the management of infective endocarditis has been the delineation of laboratory procedures that are sensitive, reliable indicators of therapeutic efficacy. Because circulating, complement-containing immune complexes of the IgG type (CICs) have been demonstrated in most humans with infective endocarditis, serum CIC levels during the natural course of the infection and in response to penicillin therapy were studied in 42 rabbits with right-sided endocarditis due to Streptococcus salivarius. A significant rise in the level of CICs in both 21 control rabbits and 21 treated rabbits was observed after induction but before treatment of infective endocarditis (P less than 0.01). In the 17 successfully treated rabbits, CIC levels fell sharply during the first week of therapy and remained at preinduction levels thereafter (P less than 0.005). In contrast, CIC values did not change significantly either in control animals or in the four treated animals with refractory endocarditis, although in the latter animals, serum bactericidal titers remained less than or equal to 1:32. These findings suggest that serial measurements of CIC levels during antimicrobial therapy of infective endocarditis may aid in monitoring therapeutic efficacy.

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.

Sera from 517 patients with various types of malignancies were assayed for immune complexes (ICs) by the Raji cell radioimmune assay. The incidence of immune complexes in these patients ranged from 16 to 52% as compared to 19% in normal controls. Increases in tumor mass and metastatic disease were associated with high levels of circulating ICs. Immunization of melanoma patients with BCG and tumor-cell vaccine produced an increase in levels of ICs. Cancer sera contained complexes of intermediate size. Tumor antigens and IgG, presumably in the form of complexes, were identified by immunofluorescence and radiolabeled antibody techniques on the surface of Raji cells incubated in ICs-containing cancer sera.

The mRNAs encoding heavy and light chains of a hybridoma-derived monoclonal IgM kappa anti-DNA autoantibody from lupus-prone MRL/Mp-lpr/lpr mice (Ighj) have been transcribed into cDNA copies and molecularly cloned, and their complete nucleotide sequences have been determined. The mRNA for the heavy chain variable region, including leader peptide and 5' untranslated region, is transcribed from a heavy chain variable region (VH) gene closely related (and possibly allelic) to VH genes of the C57BL/6 (Ighb) nitrophenyl antibody family. The deduced amino acid sequence corresponding to the light chain variable region of this autoantibody shows extensive similarities with non-autoantibody molecules of the V kappa 1 group, suggesting a common variable gene origin. The joining segments, constant regions, and 3' untranslated regions of both the heavy and light chain mRNAs are nearly identical to corresponding sequences of non-autoantibodies from normal mice. Our findings suggest that this anti-DNA autoantibody originated from the same germline repertoire as antibodies to exogenous antigens.

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.

We have investigated in vitro the magnitude, nature, and regulation of spontaneous and mitogen-induced Ig secretion by splenic lymphocytes from several autoimmune murine strains (NZB, NZB X W, MRL/l BXSB) and appropriate, normal mice. All autoimmune strains had increased numbers of mature splenic B lymphocytes, which secreted and/or contained Ig, compared to age-matched normal strains. In NZB and NZB X W mice, the high frequency of mature B cells was apparent early in life, whereas in MRL/l and BXSB mice it was first noted shortly before the clinical onset of disease. Spleen cells from young autoimmune mice of all four strains secreted predominantly IgM, but with aging and the appearance of disease, the cells switched to IgG secretion predominantly. In contrast, spleen cells from normal mice were predominantly IgM, but with aging and the appearance of disease, the cells switched to IgG secretion predominantly. In contrast, spleen cells from normal mice were predominantly IgM secretors throughout the animals' lives. Approximately 15% of the total Ig-secreting cells in older NZB, NZB X W, and MRL mice were committed to secretion of anti-ssDNA antibodies. In both autoimmune and normal spleen cells, the B-cell population alone contained fewer secreting cells than the total lymphocyte population, indicating that T cells were required to achieve maximal levels of plaque-forming cells. Spleen cells of NZB and NZB X W mice had a greater response to lipopolysaccharide (LPS) than other autoimmune and normal strains. Responsiveness to LPS, as measured by the frequency of induced Ig-secreting cells, was considerably diminished with age and onset of disease in all autoimmune but not in normal strains. LPS-induced Ig secretion by B cells of autoimmune and normal mice was subject to regulation by splenic T cells. No significant differences were observed between concanavalin-A (Con A) stimulated spleen cells from young and older autoimmune mice and normal control strains in effectively suppressing spontaneous and LPS-induced Ig secretion. Moreover, B cells from autoimmune mice and from normal strains were equally receptive to Con A-induced suppressor signals. T cells from young and older NZB and BXSB mice added to a standard number of B cells from syngeneic young mice provided equal help in enhancing LPS-induced Ig secretion, and this help in turn was equivalent to that provided by T cells from normal mice of the same H-2 haplotype. The exception was the MRL/l strain; T cells from older animals provided considerably more help than T cells from young MRL/l or T cells from young and older H-2-compatible normal mice.

Two recently described murine strains, MRL/1 and BXSB, develop a lupus-like syndrome resulting in a 50% mortality by the fifth month of age. Comparison of the immunopathological and virological characteristics of these mice with those of the NZB/NZW F1 mouse reveals several pathogenetic common denominators but no obvious common etiologic factors. In all three kinds of mice, the lupus-like syndrome consists of a fatal immune complex type glomerulonephritis and complete or near complete thymic cortical atrophy plus lymphoid hyperplasia that varies in degree among the three kinds of mice. The nephritic glomeruli contain a concentration of antinuclear antibodies plus varying amounts of stainable gp70. This syndrome is consistently correlated with abnormally elevated serum IgG levels, antinuclear antibodies, anti ds- and ssDNA antibodies, and circulating immune complexes, as well as depressed serum hemolytic complement. Features that differ among the three kinds of mice include: H2 type, anti-lymphocyte antibody, cryoglobulins, T-B cell ratios, sex incidence of disease, vasculitis, and oncornaviral flora. The serum gp70 levels in the three mice also differ considerably, but all are within the range of gp70 levels found in some immunologically normal strains.

The frequencies and absolute numbers of B and T cells in the lymphoid organs of five murine strains (NZB, (NZB X NZW)F1, BXSB, MRL/l, and MRL/n) with SLE-like syndromes were examined. We assessed the frequencies of cells bearing surface Ig, C3d and IgG Fc receptors, and theta-antigen. The sequential expression of Ig isotopes on developing B cells and the Ig isotypes expressed on adult B cells were ascertained. In addition, the Ly subsets and the expression of Ia antigens coded for by the I-J subregion of the mouse H-2 complex were examined. Compared to normal, older mice, New Zealand mice had low frequencies and absolute numbers of B cells, BXSB mice had a moderate B-cell proliferation, and MRL/l mice had normal absolute numbers of B cells but a reduced frequency concomitant with a massive T-cell proliferation. Old New Zealand mice and BXSB mice had reduced frequencies and absolute numbers of T cells compared to old controls. The developmental Ig-isotype diversity during the 1st wk of age was similar in normal mice and those with autoimmune manifestations. Mature B cells were present in lymphoid organs of New Zealand mice and BXSB mice as evidenced by the high frequency of C3d receptor-bearing cells and Ig-isotype expression (high ratio of IgM- to IgD-bearing cells) in adult spleen cells. Numbers of IgG Fc receptor-bearing cells were reduced in autoimmune mice with advanced age and disease. The proliferating T cells in MRL/l mice were found to be theta-antigen positive but Ly null. These theta+-, Ly null cells may have arisen from Ly123+ T cells. MRL/l and BXSB mice seemed normal in their content of T cells bearing Ia antigens coded for by the I-J subregion of H-2. Overall, mice with autoimmune manifestations appear to express perturbations in T and B cells with development of disease, and their patterns of change vary from one strain to another.

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.

B cell hyperactivity, a feature common to all lupus-prone murine strains, may be caused by hyperresponsiveness to, overproduction of, or bypassing of certain signals required for B cell activation, proliferation, and differentiation. In this study, we have compared the responses of B cells from three lupus-prone strains of mice (BXSB males, MRL and NZB/W females) and normal strains in a number of assays for which two or more signals are required to obtain a response. In medium to low density cultures of B cells from BXSB and NZB/W but not MRL/l lupus mice, the cells' proliferation induced by bacterial lipopolysaccharide (LPS) or anti-mu antibody was much higher than that of B cells from normal controls. At low B cell density, polyclonal activation by these substances and subsequent Ig secretion were dependent on accessory signals present in supernatants of concanavalin A-treated normal lymphocytes (CAS) or on the MRL/l proliferating T cell-derived B cell differentiation factor (L-BCDF) in both lupus-prone and immunologically normal mice. However, the responses of B cells from BXSB and NZB/W, but not MRL/l, mice to these accessory signals were higher than those of normal mice. Ig synthesis by fresh B cells of BXSB and NZB/W mice cultured in the absence of mitogens but in the presence of CAS or L-BCDF was higher than by similar cells from other strains, suggesting an increased frequency of B cells activated in vivo in these two autoimmune strains of mice. The patterns of IgG subclass secretion in response to LPS (without added CAS or L-BCDF) were abnormal in all lupus strains, with a predominance of IgG2b and/or IgG2a and low levels of IgG3, contrary to normal B cells for which IgG3 synthesis predominated. However, IgG1 synthesis in vitro by autoimmune and normal B cells alike was highly dependent on T cell-derived soluble mediators. Antigen-specific responses to SRBC in vitro of B cells from all lupus strains, like those of B cells from normal strains, required a minimum of three signals (antigen, LPS, T cell-derived antigen nonspecific helper factors). Yet, once triggered, B cells of BXSB and NZB/W mice gave higher responses than those of the other strains. We conclude that B cells of lupus mice have signal requirements similar to those of normal mice. Nevertheless, B cells of BXSB and NZB/W, but not MRL/l, lupus mice hyperrespond or process some accessory signals abnormally.

Natural thymocytotoxic autoantibodies (NTA) were found in all mouse strains. Among those strains that show autoimmune syndromes resembling human systemic lupus erythematosus (SLE), the NZB and NZB×NZW had high levels of NTA, the BXSB had moderate levels, and the MRL/1 and MRL/n had very low levels. In addition, some normal strains had high levels, sometimes even higher than the autoimmune strains. The NTA were mostly IgM and were present, but not concentrated, in the cryoprecipitates of the autoimmune mouse strains. In most strains, they were directed toward an antigen shared by thymocytes and brain. The failure to find high levels of NTA in all autoimmune mouse strains, as well as the finding of very high levels in some normal strains, make it unlikely that such autoantibodies are a fundamental etiologic factor in all murine SLE.

The participation of immune processes in the production of glomerular injury is generally accepted. The ability of the immunopathologist to readily identify and differentiate various immune pathways of injury has provided much needed insight into the mechanisms of glomerular injury and, in some cases, its etiology; it also has the potential of leading to “immunospecific” therapy. More and more, nephrology, transplantation and pathology programs are realizing the importance of immunopathologic classification of all renal disease. This is reflected in the ever increasing demand for immunopathologic services provided by groups like our own (Table 1). In this editorial we will explore the usefulness of some techniques available for detecting immune glomerular and tubular injury and see how the application of these techniques can help us identify, understand and follow the immunopathologic processes leading to renal damage.

A single intraperitoneal injection of bacterial lipopolysaccharide (LPS) or its lipid A component induced high levels of glycoprotein, gp70, in sera of several strains of mice within 24 h. This serum gp70 response induced by LPS was independent of the activation of B cells and the presence of T cells. However, serological and immunohistochemical studies demonstrated the production of gp70 by hepatic parenchymal cells and its subsequent release into the circulating blood. The expression of gp70 in the serum was enhanced not only by LPS but also other inducers of acute phase reactants (APR) such as turpentine oil or polyriboinosinic-polyribocytidylic acid. Further, the serum gp70 response was kinetically identical to those of APR. These results strongly suggest that (a) the liver may be the major source for serum gp70, (b) serum gp70 behaves like an APR, (c) its expression may be controlled by a mechanism similar to that for other APR, and (d) this glycoprotein apparently behaves as a normal host constituent and not a product of a viral genome.

Early in life, mice of four kinds [NZB, (NZB × NZW)F1, MRL/1, and male BXSB] with autoimmune disease spontaneously produced far more (>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, C3H, C57BL/6, DBA/1J, DBA/2J, LB/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.

There are now three distinct strains of “lupus mice.” Studies of these animals have provided insights not only into SLE itself but into autoimmune disease in general, and perhaps also into rheumatoid arthritis, coronary artery disease, and myocardial infarction. With all strains, it has been possible to identify specific accelerating factors that appear to contribute to early and more severe disease manifestations.

Hemopoietic cells have been reciprocally transferred between two lines of mice (MRL lpr/lpr and MRL +/+) that are congenic, differing only at the lpr (lymphoproliferation) and possibly closely linked genes. The lpr strain develops a significantly more severe and fast-paced lupus-like syndrome than +/+ strain, along with a substantially larger lymphoid mass. The results showed that: (a) hemopoietic cells of such mice were sufficient to induce the respective disease phenotypes in lethally irradiated syngeneic recipients; (b) cells of MRL +/+ mice maturing in an MRL lpr/lpr environment essentially retained the disease-producing characteristics of the donor, i.e., they induced late-life lupus without lymphadenopathy; but (c) MRL lpr/lpr cells transferred into irradiated MRL +/+ recipients unexpectedly failed to induce the early-life severe lupus and lymphoid hyperplasia of the donor, instead they caused a severe wasting syndrome resembling, in many respects, graft-vs.-host disease (GVHD). This GVHD-like syndrome developed after transfer of MRL lpr/lpr fetal liver, bone marrow, or spleen cells, and was not abrogated by elimination of T cells from the inocula. Thymectomy of the MRL +/+ recipients retarded, but did not prevent, the wasting disease. The unidirectional nature of this disease suggests that the lpr mutation conferred either a structural or regulatory defect that interfered, blocked, or altered the expression or structure of certain lymphocyte antigen(s). As a result, the MRL +/+ cells that did express this antigen(s) were recognized as foreign, and stimulated a graft-vs.-host reaction. These findings may allow definition of a new kind of rejection phenomenon caused by non-H-2 products, and may extend our understanding of the means by which the lpr gene adversely affects lymphocyte regulation and homeostasis.

Immunization of DBA/1 mice with native chick type II collagen resulted in development of polyarthritis 4-5 wk later. Sera of these mice contained high levels of anticollagen antibodies, and immunoglobulin concentrates of their sera transferred arthritis to unimmunized recipients. Histopathologically, this passively transferred arthritis resembled the early disease of immunized donors. Immunofluorescence studies demonstrated the deposition of IgG and C3 on the articular surface but not in synovial tissue of arthritic joints. Transferred, isotopically labeled anticollagen antibodies rapidly localized to the limbs and to other cartilage-containing tissues. When transfer concentrate was administered to arthritis-resistant strains, they also developed arthritis. Indeed, immunoglobulin concentrates from rats with collagen-induced arthritis transferred arthritis to naive mice. The amount of concentrate required for transfer to B10.D2 resistant mice was reduced by immunizing them with collagen 4 wk before transfer. Although susceptibility to arthritis from immunization is H-2 linked, these studies clearly demonstrate that passive transfer of arthritis depends upon injection of specific antibody and not on other host factors.

We compared spleen cells from immunologically normal mice to those from five autoimmune murine strains (NZB, NZB × W, BXSB, MRL/l, MRL/n) in the cell-mediated lympholytic response in vitro to spleen cells from syngeneic, allogeneic but H-2 identical and H-2 incompatible mice. NZB spleen cells sensitized with irradiated allogeneic but H-2d identical spleen cells lysed unmodified H-2 identical cells in a strong, cross-reactive, unidirectional response, and lysed similarly, but to a lesser extent, syngeneic cells. The NZB anti-H-2d activity was mediated by T cells and was directed against both T and B target cells. Although the anti-H-2d activity is known to be directed against minor histocompatibility differences, it appears from our experiments with congenic mice that the activity is to some extent recognized in the context of the H-2 haplotype, since it was optimum when the NZB cells matched the target cells at the K or D end of H-2. NZB cells sensitized in vitro by H-2 identical cells were only minimally stimulated to proliferate; however, such cells were able to induce in vivo GVH reactions in NZB mice. Similar in vitro anti-H-2d reactivity was observed with NZB × W spleen cells. In contrast, spleen cells from BXSB, MRL/1, and MRL/n mice were not sensitized in vitro with H-2 identical cells. Spleen cells from all autoimmune murine strains incubated with irradiated syngeneic cells were devoid of any cytolytic activity against syngeneic target cells. Cells from all autoimmune strains sampled during youth exhibited a normal effector function against H-2 incompatible spleen cells, and this function declined moderately with age and appearance of disease. In contrast to all other autoimmune strains, cells from MRL/1 mice were inadequate stimulators with H-2 incompatible cells, possibly due to massive T cell lymphoproliferation seen in these mice. Although the observed anti-H-2d activity of cells from New Zealand mice (NZB, NZB × W) may play a role in the development of their disease, the possibility that such a mechanism serves as a common etiologic pathway in the development of the murine SLE-like syndromes seems unlikely since the other autoimmune strains studied were devoid of such activity.

The effect of cyclophosphamide and x-irradiation on antibody formation in two strains of mice was studied. Cyclophosphamide was found to prevent a primary response if given prior to KLH used as antigen. An established antibody response to KLH was depressed but not ablated even after 9 months of drug administration. Single weekly administered doses of irradiation were found to markedly enhance primary antibody responses at low doses of x-irradiation and suppress only at higher doses. X-irradiation markedly enhanced anti-KLH antibody formation if given after the immune response was established.

MRL/lpr/lpr (MRL/l) mice develop a lupus-like syndrome and a disease histologically and serologically similar to human rheumatoid arthritis. Their sera contain polyclonal IgM rheumatoid factors (RF) reactive with all murine IgG subclasses (frequently strongest with IgG2a) and several heterologous IgG. To examine the repertoire and epitopic specificities of these RF, we fused splenocytes from 3.5-mo-old seropositive MRL/l mice with appropriate myeloma partners and derived 1,723 hybridomas of which 23 secreted IgMRF. These monoclonal IgMRF bound to murine IgG only, not to other murine isotypes. Eight murine IgG subclass-specific clonotypes were identified. Most clones reacted with either multiple IgG subclasses or with IgG2a alone. A few clones reacted solely with IgG2b but none reacted exclusively with IgG1 or IgG3. Monoclonal IgMRF with exclusively anti-IgG2a activity exhibited allotypic specificity, reacting, with few exceptions, with a, c, and e, but not b, d, or j IgG2a allotypes. Four clonotypes could be distinguished by cross-reactivity with IgG from species other than mice. Monoclonals possessing activity against several murine subclasses cross-reacted extensively with heterologous IgG, including all human IgG subclasses without allotypic restrictions. Monoclonal IgMRF specific for murine IgG2a or 2b did not cross-react with heterologous IgG. Based on the absence of cross-reactions by IgG2a-specific monoclonal autoantibodies, certain peptides of the IgG CH2 and CH3 domains appear to generate the antigenic determinants of the anti-IgG2a RF in MRL/l mice. All of the monoclonal RF bound to Fc and, with one exception, not to Fab fragments of murine IgG. Binding of the monoclonal RF to substrate IgG was not inhibited by Clq, thus excluding the Clq-binding site at the CH2 domain as one of the responsible epitopes in the induction of MRL/l RF. mIgMRF could be categorized as strongly, weakly, or noninhibitable by protein A, which interacts with IgG molecules at or near the CH2-CH3 junction. Inhibition appears to be caused by conformational changes and/or steric shielding of certain IgG areas distant from this junction and not by identical binding sites between protein A and RF. Certain of the mIgMRF that were weakly or not at all inhibitable by protein A were found to cross-react equally well with human Fc (CH2-CH3 domains) and pFc' (CH3 domain) fragments, indicating that the binding site for these monoclonals is at the CH3 domain. Monoclonal RF were devoid of anti-double-strand DNA, anticollagen, or antipeptidoglycan pentapeptide cross-reactivity, but one of the monoclonals cross-reacted with histones, four with single-strand DNA, and one with both histones and single-strand DNA.

Cells from a human lymphoblastoid cell line (Raji), with B-cell characteristics, and having receptors for human IgG Fc, C3b, and C3d, were used in an immunofluorescence test as in vitro detectors of immune complexes in animal and human sera. By this test, as little as 200–300 ng aggregated human gamma globulin or immune complexes per ml serum could be detected. The receptors for IgG Fc on the Raji cells were shown to be inefficient in detecting aggregated human gamma globulin and binding of aggregates to these receptors was inhibited by physiologic concentrations of 7S human IgG. Enhancement of aggregated human gamma globulin binding and binding of immune complexes formed in vitro to Raji cells was observed when the receptors for complement on these cells were used. By using the receptors for complement on Raji cells, circulating immune complexes were detected in rabbits with acute serum sickness, in mice with acute lymphocytic choriomeningitis virus infection, and in humans with immune complex type glomerulonephritis. The Raji cell test may be useful in detecting complement fixing immune complexes in different disease states, in monitoring circulating complexes in patients with immune complex diseases and in identifying the antigen(s) responsible for the induction of pathogenic immune complexes in humans and animals.

Distinguishing endocarditic from nonendocarditic septicemias is prognostically and therapeutically important. One hundred two patients with both valvular and nonvalvular sepsis were studied for the presence and quantitation of circulating immune complexes. Ninety per cent of the patients with infective endocarditis versus 50 per cent of septic patients without infective endocarditis had circulating immune complex levels (p less than 0.005). Mean circulating immune complex levels in patients with infective endocarditis were significantly higher than in those without infective endocarditis, 106 +/- 18.58 microgram/ml versus 31 +/- 7.4 microgram/ml (p less than 0.005). Only three of 52 patients without infective endocarditis had circulating immune complex levels greater than 100 microgram/ml, as opposed to 16 of 50 patients with infective endocarditis (p less than 0.005). Similarly, one of 52 patients without infective endocarditis has circulating immune complex levels greater than 200 microgram/ml, as opposed to eight of 50 patients with infective endocarditis (p less than 0.05). In 92 per cent of the patients without infective endocarditis and 76 per cent of those with infective endocarditis peak circulating immune complex levels developed within 14 days after their entry into the study, often on the initial sampling. In febrile, septicemic patients with clinical symdromes nonclassic for endocarditis, measurements of serial circulating immune complex levels may be of adjunctive diagnosis importance. If circulating immune complex levels are undetectable, endocarditis would appear less likely; alternatively, levels above 100 to 200 microgram/ml would suggest a valvular rather than nonvalvular septic focus.