Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.

Genome screening in human systemic lupus erythematosus: results from a second Minnesota cohort and combined analyses of 187 sib-pair families.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance to a multitude of self-antigens. Epidemiological data suggest an important role for genes in the etiology of lupus, and previous genetic studies have implicated the HLA locus, complement genes, and low-affinity IgG (Fcgamma) receptors in SLE pathogenesis. In an effort to identify new susceptibility loci for SLE, we recently reported the results of a genomewide microsatellite marker screen in 105 SLE sib-pair families. By using nonparametric methods, evidence for linkage was found in four intervals: 6p11-21 (near the HLA), 16q13, 14q21-23, and 20p12.3 (LOD scores >/=2.0), and weaker evidence in another nine regions. We now report the results of a second complete genome screen in a new cohort of 82 SLE sib-pair families. In the cohort 2 screen, the four best intervals were 7p22 (LOD score 2.87), 7q21 (LOD score 2.40), 10p13 (LOD score 2.24), and 7q36 (LOD score 2.15). Eight additional intervals were identified with LOD scores in the range 1.00-1.67. A combined analysis of MN cohorts 1 and 2 (187 sib-pair families) showed that markers in 6p11-p21 (D6S426, LOD score 4.19) and 16q13 (D16S415, LOD score 3.85) met the criteria for significant linkage. Three intervals (2p15, 7q36, and 1q42) had LOD scores in the range 1.92-2.06, and another 13 intervals had LOD scores in the range of 1.00-1.78 in the combined sample. These data, together with other available gene mapping results in SLE, are beginning to allow a prioritization of genomic intervals for gene discovery efforts in human SLE.

Molecular mechanisms of coxsackievirus persistence in chronic inflammatory myopathy: viral RNA persists through formation of a double-stranded complex without associated genomic mutations or evolution.

Enterovirus infection and persistence have been implicated in the pathogenesis of certain chronic muscle diseases. In vitro studies suggest that persistent enteroviruses mutate, evolving into forms that are less lytic and display altered tropism, but it is less clear whether these mechanisms operate in vivo. In this study, persistent coxsackievirus RNA from the muscle of mice afflicted with chronic inflammatory myopathy (CIM) was characterized and compared with RNA from a virus that had established a persistent infection of G8 mouse myoblasts for 30 passages in vitro. Competitive strand-specific reverse transcription-PCR and susceptibility to RNase I treatment revealed that plus- and minus-strand viral RNAs were present at nearly equivalent levels in muscle and that they persisted in a double-stranded conformation. All regions of the viral genome persisted and were amplified as a series of seven overlapping fragments. Restriction endonuclease fingerprinting coupled with sequencing indicated that there was no evolution of the viral genome associated with its persistence in muscle. This contrasted with the productive persistent infection that was established in myoblast cultures, where plus-strand RNA predominated and persistent virus developed distinct mutations. In vitro persistence proceeded by a carrier culture mechanism and was completely dependent on production of infectious virus, since persistent viral RNA was not detected in cultures subjected to antibody-mediated curing. These experiments demonstrate that persistence of coxsackievirus RNA in muscle is not facilitated by distinct genetic changes in the virus that give rise to replication-defective forms but occurs primarily through production of stable double-stranded RNA that is produced as the acute viral infection resolves. The data suggest a mechanism for coxsackievirus persistence in myofibers and perhaps other nondividing cells whereby cells that survive infection could harbor persistent viral RNA for extended times without producing detectable levels of infectious virus.

A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families.

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [D6S257, logarithm of odds (lod) = 3.90, P = 0.000011] and at three additional regions: 16q13 (D16S415, lod = 3.64, P = 0.000022), 14q21-23 (D14S276, lod = 2.81, P = 0.00016), and 20p12 (D20S186, lod = 2.62, P = 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21-33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores >/=1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.

The role of the clinical rheumatologist in the establishment of a large sibling pair resource for systemic lupus erythematosus.

OBJECTIVE: To recruit a large cohort of sibling pairs with systemic lupus erythematosus (SLE) as a clinical and biologic resource for genetic studies in SLE. METHODS: Complementary approaches were used to identify suitable families. The study was advertised in the newsletters of the Lupus Foundation of America and the Arthritis Foundation. Fliers were mailed to 250 clinical rheumatologists across the US, as well as to the local branches of the Lupus Foundation. All advertisements displayed a toll-free telephone number for interested patients to contact our group. Patients were then screened in a telephone interview by a university rheumatologist and their diagnosis was subsequently verified by telephone with the treating physician. Retrospective review of medical records was used to confirm the accuracy of the clinical data obtained by telephone interview. RESULTS: About 1400 subjects were screened by telephone over a 3 year period. After interviews with subjects and their physicians, 179 families were recruited in which at least 2 siblings have definite SLE. Based on the telephone interviews, a detailed clinical, demographic, and family history database was established for all patients in the study. Over 80% of the study subjects receive their SLE care from rheumatologists in clinical practice. CONCLUSION: We found that rheumatologists were reliable in confirming or excluding the diagnosis of SLE by telephone. Targeted patient advertising followed by physician-to-physician interviews is a time efficient and accurate method for recruiting patients with SLE for large genetic studies and may be applicable to the study of other rheumatologic conditions.

Coxsackievirus-induced chronic inflammatory myopathy: virus variants distinguish between acute cytopathic effects and pathogenesis of chronic disease.

Infection with the Tucson strain of coxsackievirus B1 (CVB1T) causes the development of chronic inflammatory myopathy (CIM) and hind limb weakness in susceptible strains of mice. In this study, a panel of six plaque-purified viruses exhibiting either small or large plaque phenotypes was derived from parental CVB1T and parental CVB1T that had been passaged through monkey kidney cells. All six variants caused similar acute histopathology in muscle, but three of four passaged viruses (AMP1, AMP2, and AMP3) did not induce CIM while the fourth (MP3) caused some hind limb weakness but without associated muscle inflammation. In contrast, both viruses (MP1 and MP2) isolated directly from the parental CVB1T stock were myopathic. Large plaque MP2 caused higher mortality and more rapid inhibition of host cell biosynthesis, but both MP1 and MP2 induced CIM that was comparable to that induced by parental CVB1T. Plaque size was a stable characteristic of the variants but did not correlate with their ability to induce CIM. Five of the six variants showed equivalent levels of replication in muscle, monkey kidney cells, and GB myoblasts while one, AMP3, was selectively impaired for replication. Receptor binding and virus-induced inhibition of host cell transcription and translation were not linked to myopathogenicity. Thus, most of the passaged variants are robust infectious viruses, suggesting that viral induction of CIM does not depend solely on cytopathogenicity during the acute infection.

Genetic determinants of susceptibility to coxsackievirus B1-induced chronic inflammatory myopathy: effects of host background and major histocompatibility complex genes.

Infection of outbred CD-1 mice with the Tucson strain of coxsackievirus B1 (CVB1T) leads to the development of chronic hind limb weakness and associated inflammatory muscle disease. Host factors that influence susceptibility have not been studied in this mouse model of chronic inflammatory myopathy (IM). Therefore, the pathogenesis was examined by using different inbred strains of mice. Initially, seven strains of mice with either the H-2d or H-2b major histocompatibility complex (MHC) haplotype were evaluated. All strains showed similar levels of acute mortality caused by viral infection, but chronic weakness or inflammation did not develop in two strains with the B6 background, regardless of their MHC haplotype. In susceptible mice, weakness was more likely to develop in the H-2d strains than in mice with the H-2b haplotype. Based on these results, H-2 congenic strains of the susceptible B10 background (C57BL/10 and B10.D2) and the resistant B6 background (C57BL/6 and B6.C-H2d) were examined in greater detail. During acute infection, the kinetics and degree of viral replication in hind limb muscle were similar among B6 and B10 strains. By 4 weeks after infection, more intense chronic muscle inflammation and pathology were observed in susceptible B10 mice of the H-2d haplotype than in those of the H-2b haplotype. Resistant B6 mice did not show signs of inflammation or calcification, but they did exhibit some myopathic features, including centralized nuclei and variations in myofiber size and shape. These changes were less common in resistant B6 mice than in B10 strains but were significant when compared with changes in uninfected controls. Viral RNA persistence and elevated titers of antiviral IgG were more prevalent in but not restricted to susceptible strains. These studies demonstrate that host background genes confer resistance to chronic IM but also that MHC genes influence disease severity. They also reveal that susceptibility to acute CVB1T infection is under different genetic control than that which mediates development of chronic post-viral IM.

Abnormal complement-mediated immune clearance in MRL-lpr/+ heterozygote mice: dose-dependent effect of the Fas antigen mutation.

We have previously demonstrated decreased complement-mediated clearance of IgG-opsonized erythrocytes in mice homozygous for the lpr mutation of the fas gene (BALB/c-lpr/lpr, C57BL/6-lpr/lpr, and MRL-lpr/lpr). To further test the hypothesis that the lpr mutation leads to a series of events resulting in selectively decreased complement-mediated immune clearance, in vivo clearance rate data were obtained from MRL-lpr/+F1, F2, and reciprocal backcross mice. Southern analysis of genomic DNA extracted from F2 and backcross mice was used to correlate the presence of the normal fas gene or the lpr mutation with normal or decreased complement-mediated clearance, respectively. Mean clearance rate constants for complement-dependent sequestration and phagocytosis were significantly decreased in the group of F1, F2, and backcross mice compared to control BALB/c mice (P < 0.0001). Data correlating clearance rate parameters from F2 and backcross mice with their respective genotype demonstrated a dose effect of the lpr mutation on abnormal complement-dependent sequestration and phagocytosis (r > 0.98), with heterozygote mice expressing mean values approximately half of those observed in +/+ homozygotes. These data demonstrate that the presence of the lpr mutation of the fas gene is strongly correlated in a dose-dependent manner with abnormal complement-mediated immune clearance. This clearance defect may be one mechanism through which the lpr mutation acts as an enhancer of autoimmune disease. Reduced clearance of immune complexes would increase the likelihood of tissue deposition and immune-mediated damage.

Duration of virus persistence and its relationship to inflammation in the chronic phase of coxsackievirus B1-induced murine polymyositis.

Mice infected with the Tucson strain of coxsackievirus B1 (CVB1T) develop chronic T cell-mediated polymyositis that is manifest as the acute infection resolves and is characterized by hindquarter weakness and muscle inflammation. This model system was used to study persistence of CVB1T RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR). For the most part, RNA persistence reflected the myotropic and neurotropic nature of the virus. At 1 month after infection, infectious virus was not detected in muscle, but persistent viral RNA was found in both skeletal and cardiac muscle, brain, and spinal cord. The kidney was weakly positive for viral RNA, whereas the liver and spleen were negative. Hindquarter muscle was assayed for persistent viral RNA at 1, 3, 6, 9, and 12 months after infection. In a few cases, persistent viral RNA was detected as late as 12 months after infection. The incidence of persistent viral RNA was high at 1 month after infection and gradually declined until, at 6 months and beyond, it was maintained in 3% to 12% of the muscles tested. Long-term viral RNA persistence was not more common in severely weak animals. However, the degree of hindquarter weakness that developed by 1 month was static thereafter and did not change over the 12-month study period. In contrast, separate experiments revealed that typical mononuclear cell (MNC) infiltration of muscle followed a time course similar to that of viral RNA persistence, peaking at 1 month and gradually resolving by 6 months. Infiltrating polymorphonuclear leukocytes (PMNs) and mast cells were present at 3 to 12 months after infection, signifying that some inflammatory activity remained. Other signs of myopathy that persisted for 12 months included a lack of muscle regeneration, variations in fiber size, and myofiber atrophy with increased perimysial and endomysial connective tissue. These results demonstrate that coxsackievirus RNA can persist in muscle for extended periods of time and are compatible with the idea that persistent virus is involved in maintaining the chronic MNC inflammation observed in murine polymyositis.

Selective, prolonged alteration of complement-mediated immune clearance after acute exposure of mice to ethanol.

A selective and prolonged alteration in complement-mediated immune clearance was found in mice given a single intraperitoneal injection of ethanol. Rate constants for the separate components of complement- and IgG Fc gamma-mediated clearance were determined using a branched series, first-order reaction sequence model and measurements of the disappearance of radiolabeled IgG-opsonized murine erythrocytes from the circulation of BALB/c mice. The rate constant governing immune clearance mediated by IgG Fc gamma receptors (k3) decreased to 16% of control at 1 hr after ethanol injection but returned to normal in 72 hr. A > 50% decrease in complement-mediated clearance occurred, with a nadir of complement-mediated sequestration (k1) and complement-dependent phagocytosis (k4) at 1 hr (P < 0.001). In this case, however, k1 and k4 rate constant values did not return to control levels until 6 weeks after the injection of ethanol. The rate constant governing C3b deactivation and release of deactivated, sensitized cells back to the circulation before they undergo phagocytosis (k2) was initially normal, but decreased in Week 6 and remained low to the end of the observation period at 22 weeks (P < 0.0001). These changes resulted in a major reduction in overall complement-mediated immune clearance up to 4 weeks after the ethanol injection. The change to normal rates for sequestration and phagocytosis coupled with decreased deactivation and release at 6 weeks postinjection resulted in a small increase in overall complement-mediated clearance that persisted through Week 22.

Murine complement-mediated immune clearance dysfunction is associated with the lymphoproliferative (lpr) gene.

Using a branched series model of immune clearance and an iterative curve fitting process, we have previously demonstrated abnormal in vivo clearance of opsonized erythrocytes in autoimmune MRL-lpr/lpr mice. This technique allows simultaneous evaluation of four rate constants governing both complement- and Fc-mediated clearance; i.e., complement-mediated sequestration (k1), C3b deactivation and release (k2), complement-dependent phagocytosis (k4), and Fc gamma-mediated sequestration and phagocytosis (k3). To evaluate genetic factors which may contribute to abnormal clearance in MRL-lpr/lpr mice, serial clearance studies were performed in congenic MRL-(+/+) mice, as well as in several mouse strains homozygous for the lymphoproliferative gene; i.e., BALB/c-lpr/lpr and C57BL/6-lpr/lpr mice. Rate data were obtained from mice 3 months through 12-18 months of age, and mean rate constant values compared to control BALB/c and/or C57BL/6 mice. Clearance of opsonized cells in MRL-lpr/lpr mice was characterized by both abnormal complement and Fc gamma receptor function with decreased complement-mediated sequestration, complement-dependent phagocytosis, and Fc gamma-mediated sequestration and phagocytosis. In contrast, abnormal clearance in congenic MRL-(+/+) mice was characterized by decreased Fc gamma-mediated sequestration and phagocytosis (P < 0.0001) only. Both complement-mediated sequestration and complement-dependent phagocytosis were significantly decreased in BALB/c-lpr/lpr and C57BL/6-lpr/lpr mice (P < 0.0001), with the pattern and magnitude of abnormal complement clearance kinetics similar to that occurring in MRL-lpr/lpr mice and contrasting with normal complement-mediated clearance in control BALB/c and C57BL/6 mice. Since decreased complement-mediated clearance was observed in three differing strains of mice homozygous for the lymphoproliferative gene, these data suggest that clearance of opsonized cells in mice is in part genetically determined, and that there may be a specific association between complement-mediated clearance dysfunction and the murine lymphoproliferative gene.

Effect of ethanol on immune clearance in mice: biphasic alteration of complement-mediated clearance with chronic ethanol ingestion.

Rate constants (k1 through k4) describing complement-mediated and Fc gamma receptor-mediated components of immune clearance were serially determined in BALB/c mice fed ethanol, 10%, in drinking water, for 24 weeks. A branched series, first order reaction sequence model of immune clearance was used to obtain the rate constants from measurements of the clearance of radiolabeled immunoglobulin G-opsonized, murine erythrocytes. A > 50% decrease in complement-mediated clearance occurred, with a nadir of complement-mediated sequestration (k1) and complement-dependent phagocytosis (k4) at 2 weeks (p < 0.003). Mean k1 and k4 rate constant values returned to control levels by week 6, and k1 increased to elevated values in weeks 10 through 20 (p < 0.05). The rate constant governing C3b deactivation and return of deactivated, sensitized cells back to the circulation (k2) was initially normal but decreased in weeks 6 through 24 (p < 0.05). Neither immunoglobulin G Fc gamma receptor-mediated clearance nor the survival of nonsensitized cells were decreased by ethanol. Mice fed ethanol had a mean blood alcohol level of 14.9 +/- 7.2 mmol/L, and their mean weight and serum complement levels did not differ from untreated controls. Complement-dependent sequestration and phagocytosis did not decrease significantly when rechallenged with 10% ethanol, but the decrease in k2 and increase in k1 did occur on rechallenge. Thus, chronic ethanol ingestion in mice is associated with an initial decrease followed by a small rebound increase in complement-mediated clearance of opsonized cells. Fc gamma receptor-mediated clearance is not decreased, and only the rebound increase in complement-mediated clearance is observed on rechallenge. This model provides a unique opportunity to study selective in vivo effects of ethanol on an important function of the immune system as well as to explore the mechanisms of ethanol tolerance in mice.

Anti-dsDNA antibodies in laboratory workers handling blood from patients with systemic lupus erythematosus.

The presence of antinuclear antibodies was determined in female laboratory workers with varying degrees of exposure to blood from patients with systemic lupus erythematosus (SLE). Subjects recruited from SLE research laboratories and a membership roster provided by the American Society for Medical Technology were classified according to self-reported frequency of handling blood from patients with SLE into high and low exposure groups. Employment and medical history were obtained by questionnaire from each study subject and their sera were tested for antibodies to double stranded DNA, single stranded DNA, and synthetic polynucleotide poly(dA-dC).poly(dG-dT) by ELISA. Analysis of the results using an independent t test showed the mean optical density for anti-dsDNA was higher in the high exposure group (mean = 0.010) than in the low exposure group (mean = 0.005, p = 0.016). There were no significant differences found between the 2 groups for anti-ssDNA or anti-poly(dA-dC).poly(dG-dT), but the means for both anti-dsDNA and anti-poly(dA-dC).poly(dG-dT) were higher in the laboratory workers than in an unexposed nonlaboratory group of women (p less than 0.001). Our results are provocative for they lend support to the hypothesis that a transmissible agent capable of causing autoantibody formation may exist in blood from patients with SLE.

Treatment of early Lyme disease.

PURPOSE: To compare the safety and efficacy of azithromycin, amoxicillin/probenecid, and doxycycline for the treatment of early Lyme disease, to identify risk factors for treatment failure, and to describe the serologic response in treated patients. PATIENTS AND METHODS: Fifty-five patients with erythema migrans and two patients with flu-like symptoms alone and fourfold changes in antibody titers to Borrelia burgdorferi were randomized to receive (1) oral azithromycin, 500 mg on the first day followed by 250 mg once a day for 4 days; (2) oral amoxicillin 500 mg and probenecid 500 mg, three times a day for each for 10 days; or (3) doxcycline, 100 mg twice a day for 10 days. If symptoms were still present at 10 days, treatment was extended with amoxicillin/probenecid or doxycycline for 10 more days. Evaluations were done at study entry and 10, 30, and 180 days later. RESULTS: Three of the patients who initially had symptoms suggestive of spread of the spirochete to the nervous system, one from each antibiotic treatment group, subsequently developed neurologic abnormalities, but symptoms in the other 54 patients resolved within 3 to 30 days after study entry. Six of the 19 patients (32%) (95% confidence interval, 13% to 57%) given amoxicillin/probenecid developed a drug eruption, whereas none of the patients given azithromycin or doxycycline had this complication. The presence of dysesthesias at study entry was the only risk factor significantly associated with treatment failure (p less than 0.001). By convalescence, 72% of the patients were seropositive, and 56% still had detectable IgM responses to the spirochete 6 months later. CONCLUSIONS: The three antibiotic regimens tested in this study were generally effective for the treatment of early Lyme disease, but the regimens differ in the frequency of side effects and in ease of administration.

Viral persistence during the developmental phase of Coxsackievirus B1-induced murine polymyositis.

Mice infected with coxsackievirus B1 (CVB1) develop a chronic hindquarter muscle weakness which resembles human polymyositis. In this study, we used in situ hybridization to screen for persistent viral RNA in hamstring and quadriceps muscles from mice that displayed various degrees of clinical weakness. At 28 to 31 days postinfection, when chronic myositis is well developed but infectious virus can no longer be recovered, persistent CVB1 RNA was found in hindquarter skeletal muscle of all 12 infected animals examined. Persistent CVB1 showed a multifocal distribution within muscle and was associated with three different histopathology patterns (HPPs). These three HPPs (HPP-1, HPP-2, and HPP-3) represent potentially different stages in the mechanism of persistence. They are based on the pattern of grains, the location of hybridization signal within the muscle, and the accompanying histopathology. In HPP-1, virus persisted in nonnecrotic muscle fibers and was not directly associated with foci of inflammatory cells. HPP-2 consisted of virus contained within necrotic myocytes that were surrounded by inflammatory cells. HPP-3 was rare and showed virus inside infiltrating mononuclear cells in a region where muscle tissue had been extensively destroyed. Persistent CVB1 occurred more frequently in severely diseased animals and in tissue sections displaying intense inflammation. Moreover, HPP-2 showed a stronger association with tissue inflammation and hindquarter weakness than did HPP-1. These data demonstrate that CVB1 persists in skeletal muscle for at least 28 to 31 days postinfection and support the concept that this persistence plays a role in the development of murine polymyositis.

Arthritis due to tuberculosis, fungal infections, and parasites.

Tuberculous, fungal, and parasitic infections infect millions of people throughout the world. While other problems usually overshadow their rheumatologic manifestations, nearly all these infections can involve bone or joints and may on occasion present with rheumatologic symptoms. The classic model of these diseases presenting as chronic monoarticular arthritis is still generally valid but other presentations, such as tenosynovitis with atypical mycobacterial infections, erythema nodosum with leprosy, coccidioidomycosis and histoplasmosis, and reactive arthritis with schistosomiasis and helminthic infections, are now well established. The most dramatic change in the epidemiology of tuberculous infections in recent years is the increasing incidence in patients with the acquired immunodeficiency syndrome (AIDS). Mycobacterium avium complex infections in particular have increased dramatically and are a major problem in the later stages of AIDS. Reports of septic arthritis and tenosynovitis due to M. avium are likely to increase over the next few years.

Differential inhibition of mitogenic responsiveness by monoclonal antibodies to beta 2-microglobulin.

A panel of 10 monoclonal antibodies (MoAbs) to human beta 2-microglobulin (beta 2m) was used to evaluate the modulation of lymphocyte activation induced by different mitogenic stimuli. All 10 MoAbs inhibited proliferative responses of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and allogeneic cells in mixed lymphocyte culture (MLC), although some MoAbs were inhibitory at much lower concentrations than others. No enhancement or direct mitogenicity was observed, but at low MoAb concentrations a delayed peak response sometimes occurred. Differentiation of B cells in PWM-stimulated PBMC cultures was also inhibited as measured by reduced accumulation of supernatant IgM and IgG. Anti-beta 2m MoAb did not interfere with the binding of PHA or PWM to PBMC, and membrane mobility as judged by subsequent capping of these lectins also appeared to be normal. Furthermore, anti-beta 2m was inhibitory when added 24 hr prior to peak responsiveness, and proliferative responses to the phorbol ester PMA in combination with ionomycin were also inhibited by MoAb, indicating that membrane-mediated events were not the target of inhibition. A comparison of the inhibitory effects of anti-beta 2m MoAb on activation by different stimuli revealed that PWM and MLC responses were much more sensitive to inhibition followed by, in order of decreasing inhibition, Con A, PHA, ionomycin alone, and PMA/ionomycin. A MoAb to a monomorphic determinant of HLA-A, B, C exhibited the same inhibitory trend, suggesting that the mechanism of inhibition was the same as for anti-beta 2m MoAbs. No inhibition was observed when PBMC were stimulated by PMA alone, suggesting that the MoAbs have little effect on activation mediated by protein kinase C but may preferentially affect the calcium-dependent pathway of activation. Thus, this differential inhibition observed with different stimuli may reflect the relative contribution of class I antigens to lymphocyte activation by a particular mitogen.

Mononuclear phagocyte system dysfunction in murine SLE: abnormal clearance kinetics precede clinical disease.

Although several studies have reported abnormal immune clearance in murine models of systemic lupus erythematosus (SLE), a consistent defect in mononuclear phagocyte function in SLE-prone mice has not been described. To evaluate the mechanism(s) of immune clearance in murine SLE, we applied the technique of kinetic analysis to clearance studies of radiolabeled, immunoglobulin-sensitized red blood cells in normal BALB/c and autoimmune BXSB, MRL-lpr/lpr, New Zealand black (NZB) and New Zealand black/white (NZB/W) mice. Clearance studies were performed in 4-week-old to 18-month-old mice with a complement-fixing rabbit IgG antimouse red blood cell antibody. Four clearance rate constants governing complement- and Fc-mediated clearance function were evaluated: complement-mediated sequestration (k1), C3b deactivation and release (k2), complement-dependent phagocytosis (k4), and Fc-mediated sequestration and phagocytosis (k3). BXSB male, MRL-lpr/lpr female and male, NZB female, and NZB/W female and male mice all had significantly decreased Fc-mediated clearance function (k3) when compared with control BALB/c mice (p less than 0.0001). This defect in Fc-mediated clearance was present in all four strains of autoimmune mice by 6 months of age and preceded the onset of serologic and clinical disease activity in NZB mice. Abnormal complement-mediated clearance was detected in MRL-lpr/lpr female and male mice, NZB female, and NZB/W female and male mice, but not in BXSB mice. In MRL-lpr/lpr mice decreased complement-mediated sequestration (k1, p less than 0.0001) and complement-dependent phagocytosis (k4, p less than 0.0001) were present as early as 4 weeks of age. In contrast, the change in complement-mediated clearance in NZB and NZB/W mice was characterized by decreased C3b deactivation and release (k2, p less than 0.001) and resulted in an enhanced early phase of clearance. Decreased k2 values in New Zealand mice occurred as early as 2 months of age, preceding serologic and clinical disease activity as well as decreased Fc receptor function. These studies demonstrated an early, progressive, and uniform defect in Fc-mediated clearance in the four murine strains of SLE studied. Complement-mediated clearance, however, varied considerably in lupus-prone mice, ranging from severe impairment in MRL-lpr/lpr to normal function in BXSB and accelerated clearance in NZB and NZB/W mice. Accelerated clearance in New Zealand mice was characterized by decreased C3b deactivation and release of antibody sensitized cells, which in turn led to increased phagocytosis of sensitized cells sequestered by complement-dependent processes.