Elimination of immune complex deposits from the kidneys of autoimmune NZB/N mice administered a xeno-spleen perfusate

54 autoimmune NZB/N mice, 6 and 10 months of age, were intravenously injected 0.2 ml solution prepared during perfusion of the isolated sheep spleen with a buffer solution. Perfusion solution was injected 8 times with 2-3-day intervals. The control group of 43 mice received intravenously 0.2 ml of a buffer solution in the similar manner. After the treatment the levels of anti-DNA antibodies and circulating immune complexes were significantly decreased in the sera of mice which received the perfusion solution, as compared with the levels of control groups. Immunofluorescent studies showed a marked decrease in the number of glomerular immune complexes deposits in mice treated with perfusion solution. Six- and ten-month old mice exhibited a similar effect. The perfusion solution may be capable of eliminating the immune complexes from the blood and kidneys of autoimmune mice.     

Synthesis of prostaglandin E by peritoneal macrophages from NZB/W mice

Peritoneal macrophages from NZB/W (murine lupus) mice spontaneously produce less prostaglandin E (PGE) than peritoneal macrophages from immunologically normal mice. Reduced PGE synthesis is seen as early as 2 months of age and becomes more profound as disease progresses. It is suggested that impaired production of PGE by peritoneal macrophages from NZB/W mice may account in part for abnormal macrophage function observed in these animals.     

Studies of reticuloendothelial function in the mouse with model immune complexes. II. Serum clearance, tissue uptake, and reticuloendothelial saturation in NZB/W mice.

We have extended the findings in the accompanying paper by characterizing the serum clearance and tissue uptake of model soluble immune complexes and the saturation of the reticuloendothelial system (RES) by these complexes in normal mice and in mice with murine lupus (NZB/W F1 females). Adult NZB/W or young C3H mice were injected with radiolabeled stable site-specifically cross-linked mouse anti-DNP oligomers as model immune complexes to probe RES function. Blood clearance and uptake by liver, spleen, and kidney were unimpaired in NZB/W mice. To determine if the RES exhibits partial saturation in the NZB/W mice, we deliberately induced a state of RES blockade with heat-aggregated human gamma-glubulin (HAG). With increasing doses of HAG (1 to 4 mg/20 g body weight) both strains similarly showed a progressive increase in RES saturation as measured by reduced liver uptake of model oligomers. Recovery from saturation was complete by 120 min in both strains. At maximal liver saturation there was only a small increase in oligomer uptake by kidney or spleen, the majority of complexes remaining within the circulation. Thus, RES capacity for handling soluble model immune complexes appears unimpaired in NZB/W mice.     

Short term administration of costimulatory blockade and cyclophosphamide induces remission of systemic lupus erythematosus nephritis in NZB/W F1 mice by a mechanism downstream of renal immune complex deposition

NZB/W F(1) mice with established nephritis were treated with a single dose of cyclophosphamide with or without a 2-wk course of murine CTLA4Ig, either alone or in combination with anti-CD154. Sixty to 80% of treated mice entered remission, and remission could be reinduced following relapse. A decrease in the frequency of anti-DNA-producing B cells and activated T cells was observed in treated mice, but this effect lasted only 3-6 wk, while remissions were sustained for up to 20 wk. Light microscopy of the kidneys of mice in remission revealed less glomerular inflammation, less tubular damage, and less infiltration of inflammatory cells. By immunofluorescence, however, IgG and C3 staining of glomeruli was no different in treated mice vs controls. Since chemokines and their receptors play an important role in inflammatory cell infiltration of affected organs in autoimmune diseases, we examined chemokine expression in the kidneys. Decreases in the expression of inflammatory cytokines and chemokines were evident in mice in the early stages of remission, but these differences were no longer present in late remission. Increased expression of CXCL13 was detected in the inflammatory infiltrates of the control NZB/NZW mice. Strikingly, we could not detect any CXCL13 in the kidneys of the treated group even in late remission. These findings suggest that costimulatory blockade together with cyclophosphamide influence the activation state of renal CD11c-positive cells and therefore lead to less B and T cell infiltration and nephritis.     

Schistosoma mansoni: Anodic polysaccharide antigen in glomerular immune deposits of mice with unisexual infections

In mice infected with unisexual Schistosoma mansoni, circulating anodic antigen was detected by immunofluorescence in glomeruli of 20 out of 22 animals from 7 to 30 weeks after infection. Circulating anodic antigen was present as finely granular deposits in the mesangium. The amount of circulating anodic antigen in the glomeruli was not clearly related to the worm burden but it increased during the course of the infection. These circulating anodic antigen deposits were accompanied by deposits of immunoglobulins, sometimes found in the same precise localization, and of complement. They probably represent the antigen part of immune complexes. Circulating anodic antigen appears to be a major candidate among the antigens involved in schistosomal glomerulopathy.     

Experimental Dirofilaria immitis-associated glomerulonephritis induced in part by in situ formation of immune complexes in the glomerular capillary wall

Eight dogs were immunized with an aqueous-soluble extract of adult Dirofilaria immitis. Subsequent to at least 7-fold increases in antibody titer, the left renal artery of each dog was infused with 6 mg of D. immitis antigen. Fourteen days after infusion, the left kidney was compared to the right kidney and preinfusion biopsies. All dogs developed glomerular lesions in the left kidney characterized by 1 or more of the following: mesangial cell proliferation, neutrophil infiltration, increased periodic acid-Schiff-positive staining of the mesangium and glomerular basement membrane (GBM), fibrin deposition, and thickening of the GBM. Left kidney glomerular immunofluorescence was positive in 7 of the 8 dogs using polyclonal antisera for canine IgG and C3 in a linear or fine granular pattern. Ultrastructural lesions were present in the left kidney of all dogs and consisted of irregular GBM thickening, intramembranous and mesangial electron-dense deposits, and mesangial and endothelial cell proliferation. Antibodies directed against D. immitis antigen were demonstrated in all kidney eluates from the left kidney. The right kidneys of 3 of the dogs developed lesions; however, in comparison to the left kidney, the lesions in the right kidneys were inconsistent, mild, and focal. The histologic findings in the left kidney were similar to those observed in dogs with naturally occurring D. immitis infections. In sham-immunized control dogs, renal arterial infusion of D. immitis antigen did not cause consistent immune complex glomerulonephritis; however, antigen adherence to glomerular capillary walls was observed by immunofluorescent microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal modulation of responses to thymus-independent and thymus-dependent antigens in autoimmune NZB/W mice

Previous work suggested that gonadal steroids influence immunity through the thymus, but the mechanisms were unclear. To investigate the effects of these hormones on immune responses to T1 and TD antigens in autoimmune mice, we studied hybrid NZB/W mice and the nonautoimmune DBA/2 strain. Mice castrated at 14 days of age were implanted with Silastic capsules releasing, in adults, physiologic levels of E2 in males or Te in females. Sham-operated controls received empty capsules. Splenic PFC were quantified 4 to 5 days after challenge with the TI2 antigen TNP-Ficoll, the TI1 antigen TNP-LPS, or the TD antigen SRBC. Young castrated NZB/W males implanted with E2 had striking enhancement of IgM responses to TNP-Ficoll when compared to castrated Te-treated females and comparable sham-operated controls of both sexes. E2 also stimulated responses to TNP-LPS. In response to challenge with SRBC, young E2-treated NZB/W males had a consistent trend to increased IgM PFC, and the stimulatory effect of E2 on IgG plaques was variable. Physiologic doses of Te had no consistent effect on responses in young mice. In old female NZB/W mice, Te caused PFC response after immunization with TNP-Ficoll to resemble age-matched NZB/W males. As sham-operated NZB/W females grew older, PFC responses to SRBC fell. This age-related phenomenon was delayed, however, in female castrates implanted with Te. In contrast, Te clearly suppressed responses to TNP-LPS. Implantation of E2 did not alter responses to TNP-Ficoll, TNP-LPS, or SRBC in nonautoimmune DBA/2 males. This finding suggested that exogenous E2 given in physiologic doses did not influence immunologic responsiveness in a normal strain to the degree seen in hormone-sensitive NZB/W mice. It was concluded that E2 enhanced responses to a variety of exogenous antigens in autoimmune NZB/W mice. The most consistent E2-induced increase in PFC response was observed with TI antigens, suggesting that E2 exerted its effects on B cells or Ts.     

Deletion of the miR-25/93/106b cluster induces glomerular deposition of immune complexes and renal fibrosis in mice

IgA nephropathy (IgAN), the most common form of primary glomerulonephritis, is caused by immune system dysfunction and affects only the kidneys. miRNA was involved in IgAN, in which their roles are still unknown. Herein, we found increased glomerular medulla size, proteinuria, kidney artery resistance, kidney fibrosis and immune complex deposition in 5-month miR-25/93/106b cluster knockout (miR-TKO) mice. In vitro, the inhibition of miR-25 cluster could promote cell proliferation and increase fibrosis-related protein and transferrin receptor (TFRC) expression in human renal glomerular mesangial cell (HRMC). Luciferase assay revealed that inhibition of miR-93/106b cluster could upregulate Ccnd1 expression through direct binding with the 3’UTR of Ccnd1. Conversely, inhibition of Ccnd1 expression prevented miR-93/106b-induced effect in HRMC. These findings suggested that miR-25 cluster played an important role in the progression of IgAN, which provided new insights into the pathogenesis and treatment of IgAN.     

Regulation of immune function by calorie restriction and cyclophosphamide treatment in lupus-prone NZB/NZW F1 mice

We compared the effects of calorie restriction (CR) and cyclophosphamide (CTX) on the progression of lupus nephritis and immunological changes in NZB/NZW F1 mice. Ad libitum (AL)/CTX and CR delayed onset of proteinuria and significantly decreased serum levels of anti-dsDNA, anti-histone, and circulating immune complex antibodies. CTX and CR prevented the increase in and activation of B cells, the decline in CD8(+) T cells, and maintained a higher proportion of na?ve CD4(+) and CD8(+) cells. MHC class I antigen and LFA-1 expression on CD8(+) T cells and MHC class II antigen on B cells were also decreased. AL/CTX and CR prevented the increase in production of IL-10 and up-regulated IL-2 production in T cells ex vivo. We concluded that both CR and CTX can delay the onset of autoimmune disease, in part by maintaining higher numbers of na?ve T cells and the immune responsiveness of T cells and decreasing the proportion of B cells.     

Binding of model immune complexes to erythrocyte CR1 facilitates immune complex uptake by U937 cells

The E C3b/C4b receptor (CR1) has been shown to rapidly bind large complement-fixing immune complexes (IC) both in vivo and in vitro. It has been proposed that E (RBC) CR1 act as a shuttle mechanism, binding circulating IC and transporting them to tissue macrophages, thereby preventing their deposition in target tissues. In this study we have established an in vitro model system with which to study the transfer of model IC from CR1 on the RBC surface to phagocytic cells. Aggregated IgG (AHG) was opsonized with C3b, bound to RBC CR1, and the binding of these RBC-bound IC by a human monocyte cell line (U937 cells) was examined. U937 binding of AHG from the RBC surface was complete within 2 min, whereas binding of the same AHG from solution required 30 to 60 min. Despite the difference in kinetics of binding, the total amount of IC bound by U937 cells at equilibrium was the same for RBC-bound AHG and for AHG in solution. The transfer of AHG from the RBC to the U937 cell did not require exogenous factor I and was not accompanied by binding of RBC to U937 cells or by erythrophagocytosis. Our data lend support to the hypothesis that binding of IC to RBC CR1 may facilitate the clearance of IC from the circulation by enhancing their uptake by phagocytic cells.     

Regulation of an in vitro anti-DNA antibody response by thymocytes and T cells from NZB/W and normal mice

The spontaneous in vitro anti-DNA antibody response generated by preautoimmune and many normal mouse spleen cells was suppressed by the addition of syngeneic thymocytes or splenic T cells. Suppressive activity was found in normal mice (DBA/2J) and to an equivalent degree in the autoimmune (New Zealand Black X New Zealand White)F1 (B/W) strain. The suppressor cells were cortisone-resistant, radiosensitive and carried Lyt 1 and Lyt 2 markers. Nonspecific suppression was not involved since the primary and primed in vitro anti-sheep erythrocyte (anti-SRBC) responses were unaffected. Both spontaneous and lipopolysaccharide-stimulated anti-DNA antibody responses could be suppressed. There was no difference in the suppressive activity of cells from young or old, normal or autoimmune mice. These T cells may therefore play a role in preventing the anti-DNA antibody response in normal and young B/W mice, but evidently fail to influence the development of in vivo anti-DNA autoimmune responses in the old B/W mice.     

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/wf1 mice.

Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF₁ mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF₁ mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF₁ mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF₁ recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF₁ mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.     

Pulmonary immune responses induced in BALB/c mice by Paracoccidioides brasiliensis conidia

Knowledge concerning the host–Paracoccidioides brasiliensis interactions is abundant. Yet, most of the experimental studies have used yeast cells to prepare the corresponding inoculum. As these cells do not represent the naturally infecting propagules, the corresponding experiments by-pass the earlier stages of such interactions. Studies done in patients, who also harbour yeast cells, suffer from the same bias. The review presented below focuses on the immune responses of BALB/c mice infected with conidia obtained from P. brasiliensis mycelial form cultures, the fungal stage most probably existing in nature. As such, the corresponding experiments would copy the onset and course of the human infection. A large number of experimental studies done by the CIB Medical and Experimental Mycology Unit in a period of almost 25 years have been revised and extracted so as to present a comprehensive record on the immune responses induced when mice are infected intranasally with the conidia. The establishment of this mouse model has permitted the analysis of the immune responses taking place during the early and late stages post-challenge. This unique model has made possible to characterize the course of the experimental disease including the inflammatory reaction, the expression of cytokines and of the various molecules associated to these responses, all of which lead to granuloma formation. The latter structure serves as a nest for the development of fibrosis. Thus, we have also obtained a glimpse on the complexity that accompanies the fibrosis, the most common sequelae of paracoccidioidomycosis. Additionally, a concerted effort has been made to appraise the whole gamut of immune factors and related molecules that directly or indirectly, contribute to shape the pathogenesis of this Latin American mycosis.     

Beneficial effects of treatment with transglutaminase inhibitor cystamine on macrophage response in NZB/W F1 mice

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder of unknown etiology. However, the definitive mechanisms remain obscure. Recently, transglutaminase 2 (TG2) was implicated in the pathogenesis of SLE. Cystamine, which inactivates TG2 activity by forming a mixed disulfide, may interfere with and inhibit other thiol-dependent enzymes such as caspases. To investigate the effects of cystamine in SLE pathogenesis, this in vivo study assessed the serum and macrophage response after administration of cystamine to NZB/W F(1) mice. The experimental results demonstrated for the first time a significant reduction in TG2 and matrix metalloproteinase (MMP)-9 activity; tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, TG2, tumor necrosis factor alpha, and tumor growth factor beta mRNA expression; and anticardiolipin autoantibodies (aCL) in NZB/W F(1) mice following cystamine administration. It strongly suggests the therapeutic potential of cystamine in SLE.     

Hormonal manipulation of the prenatal environment alters reproductive morphology and increases longevity in autoimmune NZB/W mice

Steroid hormones, which affect development of reproductive traits, alter immune responses in rodents and appear to control severity of disease in F1 hybrid NZB/W mice, an animal model of systemic lupus erythematosus. We tested the hypothesis that exposure of NZB/W fetuses to altered hormonal environments would influence subsequent expression of autoimmune renal disease and affect longevity. NZB females, pregnant with NZB/W fetuses, were treated from Days 13-18 of gestation with testosterone or the antiandrogen, flutamide. Similar treatments were carried out in C57BL/6 dams mated to DBA/2 males to permit comparison with nonautoimmune hybrid mice. Serum concentrations of testosterone were greater in testosterone-implanted dams of both strains, but concentrations of estradiol were greater only in C57BL/6 dams treated with flutamide. Alpha fetoprotein (AFP), which binds estrogen and modulates immune responsiveness, was greater in serum from both groups of testosterone-treated dams, while flutamide treatment increased serum AFP only in NZB dams. We conclude that factors governing circulating estradiol and AFP differed in pregnant NZB and C57BL/6 females. Morphological analyses confirmed effects of hormonal manipulation on the developing fetuses. Testosterone implants resulted in female offspring with greater anogenital spaces, and treatment of dams with flutamide eliminated the expected difference between anogenital spaces in females and males. Effects of altered prenatal hormonal environments on immune-mediated disease in NZB/W offspring were examined in a longevity study. Early deaths were delayed in NZB/W females produced by flutamide-treated dams. An unexpected result was observed in NZB/W males. Male offspring from both testosterone- and flutamide-treated mothers lived longer than males from control dams. This paradox suggested that a characteristic shared by both groups of treated NZB dams had similar effects on the developing fetuses. It is proposed that elevated concentrations of AFP modulated the course of autoimmune disease and contributed to increased longevity in NZB/W offspring of treated dams.     

Humoral immune responses induced by Gymnorhynchus gigas extracts in BALB/c mice.

The aim of this study was to determine if the plerocercoid larvae of Gymnorhynchus gigas, a common cestode of the ray's bream (Brama raii), possess antigenic compounds potentially capable of provoking anaphylactic episodes. A murine experimental model, using BALB/c mice, was developed to study the humoral immune response induced by G. gigas extracts. A highly specific humoral immune response was detected and cross-reactions were not observed between parasite and host antigens. The presence of IgM and IgG3 levels suggest the presence of thymus-independent antigens in the parasitic extract. The IgG antibody class showed the highest levels, with the IgG1 the predominant subclass. These IgG1 levels are in accordance with the supposed presence of a type I allergic reaction after the ingestion of G. gigas plerocercoids parasitizing fish, as well as inducing anaphylaxia in fish. These results indicate that somatic products released from ingested larvae of G. gigas could induce the development of a Th2 response capable of causing allergic disorders.     

Evidence that autoimmunity in NZB mice is caused by a defect in immune specificity rather than a generalized defect in tolerance of self-antigens

NZB mice which were already producing anti-erythrocyte autoantibodies were not able to respond to their own liver F antigen, thus providing evidence that their autoimmunity is not caused by a generalized breakdown in self-tolerance mechanisms. The specificity of autoantibodies produced in the spontaneous hemolytic anemia was different from that of antierythrocyte antibodies induced in normal mice and in young NZB mice by injections of rat erythrocytes. This indicates that the B-cell clones which can be triggered by heterologous antigen are different from those responsible for the NZB disease; the latter clones may not exist in normal mice.     

Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.