Engraftment of peripheral blood mononuclear cells from systemic lupus erythematosus and antiphospholipid syndrome patient donors into BALB-RAG-2-/- IL-2Rγ-/- mice: a promising model for studying human disease.

OBJECTIVE: To construct a humanized mouse model of systemic lupus erythematosus (SLE) that resembles the human disease in order to define the pathophysiology and targets for treatments. METHODS: We infused peripheral blood mononuclear cells (PBMCs) from SLE patients into BALB- RAG-2-/- IL-2Rγ-/- double-knockout (DKO) mice, which lack T cells, B cells, and natural killer cells. PBMCs from 5 SLE patients and 4 normal donors were infused intravenously/intraperitoneally at a density of 3-5×10(6) cells per animal into nonirradiated 4-5-week-old mice. We evaluated the engraftment of human CD45+ cells and monitored the plasma levels of human IgG, anti-double-stranded DNA (anti-dsDNA) antibody, and anticardiolipin antibody (aCL), as well as proteinuria and kidney histology. RESULTS: There was 100% successful engraftment in 40 DKO mice infused with human PBMCs. In the PBMC fraction from SLE PBMC-infused DKO (SLE-DKO) mice and normal donor PBMC-infused DKO (ND-DKO) mice, an average of 41% and 53% human CD45+ cells, respectively, were observed at 4 weeks postengraftment, with 70-90% CD3+ cells. There were fewer CD3+CD4+ cells (mean±SEM 5.5±2.1%) and more CD3+CD8+ cells (79.4±3.6%) in the SLE-DKO mice as in the SLE patients from which the PBMCs were derived. CD19+ B cells and CD11c+ monocytic cells were found in the spleen, lung, liver, and bone marrow. There was no significant difference in plasma levels of human IgG and anti-dsDNA antibodies between SLE-DKO and ND-DKO mice. Levels of aCL were significantly higher in all SLE-DKO mice infused with PBMCs from an SLE patient who had high titers of aCL. SLE-DKO mice had proteinuria, human IgG deposits in the kidneys, and a shorter life span. In SLE-DKO mice engrafted with PBMCs from the aCL-positive patient, we found microthrombi and infiltration of CD3+, CD8+, and CD19+ cells in the glomeruli, recapitulating the human antiphospholipid syndrome in these mice. CONCLUSION: We established a novel humanized SLE-DKO mouse exhibiting many of the immunologic and clinical features of human SLE.

Systemic lupus erythematosus monocytes are less responsive to interleukin-10 in the presence of immune complexes.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by autoantibody production and immune complex deposition. The level of interleukin-10 (IL-10), predominantly an antiinflammatory cytokine, is paradoxically elevated in patients with SLE. The aim of this study was to examine the hypothesis that the antiinflammatory function of IL-10 is impaired in monocytes from patients with SLE with long-term exposure to immune complexes. METHODS: CD14+ monocytes were isolated from healthy donors and patients with SLE. Cultured CD14+ cells were treated with heat-aggregated human IgG (325 µg/ml) in the presence or absence of IL-10 (20 ng/ml). To study gene expression, RNA was extracted 3 hours after treatment. To study cytokine production, supernatants were harvested after 8 hours. To study IL-10 signaling, cell lysates were obtained from CD14+ cells treated with human IgG (325 µg/ml) for 1 hour followed by IL-10 (20 ng/ml) treatment for 10 minutes. Western blot analysis was used to assess STAT-3 phosphorylation. All experiments were performed in pairs. RESULTS: When stimulated with human IgG, SLE monocytes produced more tumor necrosis factor α (TNFα) and IL-6 than did control cells. The suppressive effect of IL-10 on human IgG-induced TNFα and IL-6 production was lower in SLE monocytes compared with control monocytes, although IL-10 receptor expression was similar in SLE and control monocytes. Human IgG suppressed IL-10 receptor expression and altered IL-10 signaling in control monocytes. Like SLE monocytes, interferon-α (IFNα)-primed control monocytes stimulated with human IgG were also less responsive to IL-10. CONCLUSION: Human IgG and IFNα modulate IL-10 function. In SLE monocytes, which are considered to be IFNα primed and persistently exposed to immune complexes, responses to IL-10 are abnormal, limiting the antiinflammatory effect of this cytokine.

Targeted inhibition of complement activation prevents features of preeclampsia in mice.

Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. In mouse models, complement activation in the placenta is associated with abnormal placental development and miscarriage, and inhibiting complement prevents fetal injury. We mated two mouse strains, DBA/2 and CBA/J, expecting that the pregnancies might show features of preeclampsia and of immunologically mediated pregnancy loss. Along with placental dysfunction, these matings resulted in proteinuria, elevated BUN, fibrin deposition, and glomerular endotheliosis. We blocked placental complement activation throughout pregnancy by administering a single dose of the C3 inhibitor CR2-Crry given on day 5 of the pregnancy. This procedure specifically targets the sites of complement activation without inducing any systemic effects. Placental complement inhibition prevented oxidative stress and placental dysfunction, as well as proteinuria and renal pathologic features of preeclampsia. Thus, local blockade of complement activation at the maternal-fetal interface rescues preeclampsia in mice, and identifies new treatments. Hence, complement triggers a feed-forward cycle of placental damage, antiangiogenic factor production, and maternal vascular damage in patients.

Infliximab treatment shifts the balance between stimulatory and inhibitory Fcgamma receptor type II isoforms on neutrophils in patients with rheumatoid arthritis.

OBJECTIVE: Human neutrophils express both activating and inhibitory Fcgamma receptors (FcgammaR), and their relative expression determines the inflammatory response to immune complexes. Tumor necrosis factor alpha (TNFalpha) up-regulates the expression of stimulatory FcgammaRIIa on neutrophils in vitro, and amplifies immune complex-induced activation of neutrophils in vivo. This study was undertaken to determine whether TNFalpha blockade in patients with rheumatoid arthritis (RA) alters the balance of activating FcgammaR and inhibitory FcgammaR and thereby decreases inflammation. METHODS: We used fluorescence-activated cell sorting and Western blotting to examine FcgammaR expression on neutrophils in 24 patients with RA, preceding their first infusion of infliximab and immediately prior to >or=3 subsequent infusions. RESULTS: In 13 of 24 patients (54.2%), there was a decrease in the expression of the predominant activating FcgammaR, FcgammaRIIa, after treatment with infliximab, an effect that persisted over >or=3 months of treatment. Although prior to initiation of infliximab therapy the inhibitory FcgammaR, FcgammaRIIb, was undetectable in neutrophils from 23 of 24 patients with RA, FcgammaRIIb protein was detected by Western blotting in 9 patients (37.5%) at the time of the third infliximab infusion. The induction of inhibitory FcgammaRIIb was always associated with decreased levels of FcgammaRIIa, and improvement following infliximab therapy, measured using the Health Assessment Questionnaire, was significantly associated with down-regulation of FcgammaRIIa. CONCLUSION: Our findings indicate that TNFalpha inhibition may reduce inflammation in patients with RA by restoring the balance of activating and inhibitory FcgammaR and thereby raising the threshold for immune complex-mediated activation of neutrophils.

Regulated expression of FcgammaR in human dendritic cells controls cross-presentation of antigen-antibody complexes.

Receptors for IgG (FcgammaR) expressed in dendritic cells (DCs) influence the initiation of Ab-mediated immunity. Dynamic variations in FcgammaR expression allow DCs to adjust their capacity to capture Ab-opsonized Ag. The current paradigm predicts a progressive decline in FcgammaR-mediated phagocytic function upon DC maturation. Surprisingly, we find that expression of the phagocytic receptor FcgammaRIIa is preserved in immature and mature DCs at comparable levels with macrophages. Moreover, phagocytosis of antigenic peptides directed to FcgammaRIIa on DCs leads to dramatic increases in Ag cross-presentation and T cell activation. In immature DCs, high expression of inhibitory FcgammaRIIb correlates with decreased uptake and cross-presentation of Ab-Ag complexes. In contrast, engagement of FcgammaRIIb is not associated with changes in cross-presentation in mature DCs. We provide evidence that FcgammaRIIb expression is patently reduced in mature DCs, an effect that is modulated by treatment with cytokines. The regulated expression of activating and inhibitory FcgammaRs in DCs emerges as a critical checkpoint in the process of Ag uptake and cross-presentation.

FcgammaRIIa is a target for modulation by TNFalpha in human neutrophils.

Activation of neutrophils by the interaction of immune complexes with Fc gamma receptors (FcgammaR) is amplified in tumor necrosis factor-alpha (TNFalpha)-primed cells, whereas interleukin-10 (IL-10) has been reported to suppress cytokine-mediated neutrophil activation. We examined whether the expression and function of FcgammaR in human neutrophils is modulated by TNFalpha and IL-10 in vitro, and whether FcgammaRIIa expression is altered following treatment with the TNFalpha inhibitor infliximab in rheumatoid arthritis (RA) patients in vivo. TNFalpha treatment induced upregulation of expression and function of the major activating Fc receptor, FcgammaRIIa, in neutrophils from healthy donors. Unexpectedly, treatment with IL-10 led to gain of FcgammaRIIa function in TNFalpha-primed neutrophils. In neutrophils from RA patients initiating infliximab therapy and followed longitudinally through consecutive treatments, FcgammaRIIa protein decreased during the course of TNFalpha blockade, indicating that FcgammaRIIa is a target of TNFalpha modulation in human neutrophils in vivo.

Decreased transcription of the human FCGR2B gene mediated by the -343 G/C promoter polymorphism and association with systemic lupus erythematosus.

The role for inhibitory Fc gamma receptors class IIb (FcgammaRIIb) in the onset, progression and severity of several animal models of autoimmune diseases is well established. By contrast, the pathogenic potential of FcgammaRIIb in human autoimmune diseases remains largely unknown. Here we report the identification of a polymorphism in the human FCGR2B promoter (dbSNP no. rs3219018) that is associated in homozygosity with systemic lupus erythematosus (SLE) phenotype in European-Americans (OR=11.1, P=0.003). Experimental evidence correlates the polymorphism (a G-C substitution at position -343 relative to the start of transcription) with altered FcgammaRIIb expression and function. The G-C substitution correlated with decreased transcription of the FCGR2B promoter, and resulted in decreased binding of the AP1 transcription complex to the mutant promoter sequence. The surface expression of FcgammaRIIb receptors was significantly reduced in activated B cells from (-343 C/C) SLE patients. These findings suggest that genetic defects may lead to deregulated expression of the FCGR2B gene in -343 C/C homozygous subjects, and may play a role in the pathogenesis of human SLE.

The CY domain of the Fcgamma RIa alpha-chain (CD64) alters gamma-chain tyrosine-based signaling and phagocytosis.

Although the cytoplasmic domain of the human FcgammaRIa alpha-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcgammaRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcgammaRIa, we developed a series of human FcgammaRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the alpha-chain of FcgammaRIa and suggest that the cytoplasmic domain of FcgammaRI regulates the different functional capacities of the FcgammaRIa-receptor complex.