IgG immune complexes with Staphylococcus aureus protein A enhance osteoclast differentiation and bone resorption by stimulating Fc receptors and TLR2.

Staphylococcus aureus is a main pathogen of osteomyelitis and protein A is a virulence factor with high affinity for IgG. In this study, we investigated whether S. aureus affects the differentiation and bone resorption of osteoclasts through the IgG-binding capacity of protein A. Staphylococcus aureus pre-treated with serum or IgG showed marked enhancement in osteoclastogenesis and bone resorption compared to non-treated S. aureus or a protein A-deficient mutant. Blocking of the Fc receptor and deletion of the Fcγ receptor gene in osteoclast precursor cells showed that enhanced osteoclastogenesis stimulated by S. aureus IgG immune complexes (ICs) was mediated by the Fc receptor on osteoclast precursor cells. In addition, osteoclastogenesis stimulated by S. aureus ICs but not the protein A-deficient mutant was markedly reduced in osteoclast precursor cells of Myd88-knockout mice. Moreover, NFATc1, Syk and NF-κB signals were necessary for osteoclastogenesis stimulated by S. aureus ICs. The results suggest the contribution of a of Toll-like receptor 2 (TLR2)-Myd88 signal to the activity of S. aureus ICs. We further examined the expression of pro-inflammatory cytokines that is known to be enhanced by FcγR-TLR cross-talk. Osteoclasts induced by S. aureus ICs showed higher expression of TNF-α and IL-1ß, and marked stimulation of proton secretion of osteoclasts activated by pro-inflammatory cytokines. Finally, injection of S. aureus, but not the protein A-deficient mutant, exacerbated bone loss in implantation and intra-peritoneal administration mouse models. Our results provide a novel mechanistic aspect of bone loss induced by S. aureus in which ICs and both Fc receptors and TLR pathways are involved.

Knockout of the neonatal Fc receptor in cultured podocytes alters IL-6 signaling and the actin cytoskeleton.

The neonatal Fc receptor (FcRn) has been shown to be required for antigen presentation in dendritic cells, and global knockout of FcRn attenuates immune-mediated kidney disease. Podocytes express interleukin-6 (IL-6) receptor and produce IL-6 under proinflammatory conditions. Here we examined the role of FcRn in the IL-6-mediated inflammatory response in podocytes. We examined IL-6 production by ELISA and expression by qPCR in wild type (WT) and FcRn knockout (KO) podocytes after treatment with proinflammatory stimuli as well as IL-6-mediated signaling via the JAK/STAT pathway. We also examined podocyte motility in cultured WT and KO podocytes after a proinflammatory challenge. We found that FcRn KO podocytes produced minimal amount of IL-6 after treatment with albumin, IgG, or immune complexes whereas WT podocytes had a robust response. FcRn KO podocytes also had minimal expression of IL-6 compared with WT. By Western blotting, there was significantly less phosphorylated STAT3 in KO podocytes after treatment with IFNγ or immune complexes. In a scratch assay, FcRn KO podocytes showed increased motility comparted KO, suggesting a defect in actin dynamics. Cultured FcRn KO podocytes also demonstrated abnormal stress fibers compared with WT and the defect could be rescued by IL-6 treatment. This study shows that in podocytes, FcRn modulates the IL-6 mediated response to proinflammatory stimuli and regulates podocytes actin structure, motility and synaptopodin expression.

Elimination of activating Fcγ receptors in spontaneous autoimmune peripheral polyneuropathy model protects from neuropathic disease.

Spontaneous autoimmune peripheral polyneuropathy (SAPP) is a reproducible mouse model of chronic inflammatory peripheral neuropathy in female non-obese diabetic mice deficient in co-stimulatory molecule, B7-2 (also known as CD86). There is evidence that SAPP is an interferon-γ, CD4+ T-cell-mediated disorder, with autoreactive T-cells and autoantibodies directed against myelin protein zero involved in its immunopathogenesis. Precise mechanisms leading to peripheral nerve system inflammation and nerve injury including demyelination in this model are not well defined. We examined the role of activating Fc-gamma receptors (FcγRs) by genetically ablating Fcγ-common chain (Fcer1g) shared by all activating FcγRs in the pathogenesis of this model. We have generated B7-2/ Fcer1g-double null animals for these studies and found that the neuropathic disease is substantially ameliorated in these animals as assessed by behavior, electrophysiology, immunocytochemistry, and morphometry. Our current studies focused on characterizing systemic and endoneurial inflammation in B7-2-null and B7-2/ Fcer1g-double nulls. We found that accumulation of endoneurial inflammatory cells was significantly attenuated in B7-2/ Fcer1g-double nulls compared to B7-2-single nulls. Whereas, systemically the frequency of CD4+ regulatory T cells and expression of immunosuppressive cytokine, IL-10, were significantly enhanced in B7-2/ Fcer1g-double nulls. Overall, these findings suggest that elimination of activating FcγRs modulate nerve injury by altering endoneurial and systemic inflammation. These observations raise the possibility of targeting activating FcγRs as a treatment strategy in acquired inflammatory demyelinating neuropathies.

B Cell-Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B Cell Immune Responses.

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells, Myd88 is required for anti-DNA and anti-RNA autoantibody responses, whereas Fcer1g is not expressed in these cells. The roles of these receptors in myeloid cells during B cell autoimmune activation remain less clear. To investigate the roles of Myd88 and Fcer1g in non-B cells, we transferred anti-self-IgG (rheumatoid factor) B cells and their physiologic target Ag, anti-chromatin Ab, into mice lacking Fcer1g, Myd88, or both and studied the extrafollicular plasmablast response. Surprisingly, we found a markedly higher and more prolonged response in the absence of either molecule; this effect was accentuated in doubly deficient recipients, with a 40-fold increase compared with wild-type recipients at day 10. This enhancement was dependent on CD40L, indicating that Myd88 and FcRγ, presumably on myeloid APCs, were required to downregulate T cell help for the extrafollicular response. To extend the generality, we then investigated a classic T cell-dependent response to (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken γ globulin and found a similar effect. Thus, these results reveal novel regulatory roles in the B cell response for receptors that are typically proinflammatory.

Crucial Role for Immune Complexes but Not FcRn in Immunization against Anti-TNF-α Antibodies after a Single Injection in Mice.

The immunogenicity of infliximab and adalimumab is a major concern because patients may develop Abs also called antidrug Abs (ADA), directed against these anti-TNF-α Abs after just a few weeks of treatment. These ADAs can lead to a decrease in biologic concentration, which is associated with lower treatment efficacy. Our aim was to study the involvement of immune complexes and neonatal Fc receptor (FcRn) in the emergence of ADAs in the case of anti-TNF-α Abs. Wild type and FcRn knockout mice were injected once with either infliximab or adalimumab, alone or preincubated with TNF-α. Adalimumab cross-reacts with murine TNF-α whereas infliximab is species specific. When injected alone, only adalimumab elicited a humoral response. By preforming immune complexes with TNF-α, an anti-infliximab response was elicited. Surprisingly, both wild type and FcRn knockout mice were able to mount an immune response against anti-TNF-α Abs, suggesting that immune complexes are a major determinant of this immunization.

Mucosal vaccination promotes clearance of Streptococcus agalactiae vaginal colonization.

Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants, and colonization of the maternal genital tract is the primary risk factor for newborn infection. Despite the importance of mucosal colonization in GBS pathogenesis, relevant host and bacterial factors are incompletely understood. We investigated the role of humoral immunity in clearance of vaginal colonization in vivo. B-cell-deficient mice or those lacking neonatal Fc-receptor, a mediator of IgG transport to the vaginal mucosa, exhibit prolonged GBS vaginal colonization compared to wild type animals. Intranasal but not intramuscular immunization induced systemic and mucosal immune responses and decreased GBS colonization duration without altering initial colonization density. Vaccine-induced clearance of GBS was serotype-specific, suggesting a role for anti-capsule antibodies in protection. Our results support a role for humoral immunity in GBS eradication from the female genital tract and suggest that mucosal vaccination may prime colonization clearance.

FcRγ-dependent immune activation initiates astrogliosis during the asymptomatic phase of Sandhoff disease model mice.

Sandhoff disease (SD) is caused by the loss of ß-hexosaminidase (Hex) enzymatic activity in lysosomes resulting from Hexb mutations. In SD patients, the Hex substrate GM2 ganglioside accumulates abnormally in neuronal cells, resulting in neuronal loss, microglial activation, and astrogliosis. Hexb-/- mice, which manifest a phenotype similar to SD, serve as animal models for examining the pathophysiology of SD. Hexb-/- mice reach ~8 weeks without obvious neurological defects; however, trembling begins at 12 weeks and is accompanied by startle reactions and increased limb tone. These symptoms gradually become severe by 16-18 weeks. Immune reactions caused by autoantibodies have been recently associated with the pathology of SD. The inhibition of immune activation may represent a novel therapeutic target for SD. Herein, SD mice (Hexb-/-) were crossed to mice lacking an activating immune receptor (FcRγ-/-) to elucidate the potential relationship between immune responses activated through SD autoantibodies and astrogliosis. Microglial activation and astrogliosis were observed in cortices of Hexb-/- mice during the asymptomatic phase, and were inhibited in Hexb-/- FcRγ-/- mice. Moreover, early astrogliosis and impaired motor coordination in Hexb-/- mice could be ameliorated by immunosuppressants, such as FTY720. Our findings demonstrate the importance of early treatment and the therapeutic effectiveness of immunosuppression in SD.

Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation.

Alzheimer's disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded protein assemblies evade detection, as they are host-derived. Here, we show that when misfolded tau assemblies enter the cell, they can be detected and neutralized via a danger response mediated by tau-associated antibodies and the cytosolic Fc receptor tripartite motif protein 21 (TRIM21). We developed fluorescent, morphology-based seeding assays that allow the formation of pathological tau aggregates to be measured in situ within 24 h in the presence of picomolar concentrations of tau seeds. We found that anti-tau antibodies accompany tau seeds into the cell, where they recruit TRIM21 shortly after entry. After binding, TRIM21 neutralizes tau seeds through the activity of the proteasome and the AAA ATPase p97/VCP in a similar manner to infectious viruses. These results establish that intracellular antiviral immunity can be redirected against host-origin endopathogens involved in neurodegeneration.

Contribution of murine IgG Fc regions to antibody binding to the capsule of Burkholderia pseudomallei.

Immunoglobulin G3 (IgG3) is the predominant IgG subclass elicited in response to polysaccharide antigens in mice. This specific subclass has been shown to crosslink its fragment crystallizable (Fc) regions following binding to multivalent polysaccharides. Crosslinking leads to increased affinity through avidity, which theoretically should lead to more effective protection against bacteria and yeast displaying capsular polysaccharides on their surface. To investigate this further we have analyzed the binding characteristics of 2 IgG monoclonal antibody (mAb) subclass families that bind to the capsular polysaccharide (CPS) of Burkholderia pseudomallei. The first subclass family originated from an IgG3 hybridoma cell line (3C5); the second family was generated from an IgG1 cell line (2A5). When the Fc region of the 3C5 IgG3 is removed by proteolytic cleavage, the resulting F(ab')2 fragments exhibit decreased affinity compared to the full-length mAb. Similarly, when the parent IgG3 mAb is subclass-switched to IgG1, IgG2b, and IgG2a, all of these subclasses exhibit decreased affinity. This decrease in affinity is not seen when the 2A5 IgG1 mAb is switched to an IgG2b or IgG2a, strongly suggesting the drop in affinity is related to the IgG3 Fc region.

Specific IgA Enhances the Transcytosis and Excretion of Hepatitis A Virus.

Hepatitis A virus (HAV) replicates in the liver, and is excreted from the body in feces. However, the mechanisms of HAV transport from hepatocytes to the gastrointestinal tract are poorly understood, mainly due to lack of suitable in vitro models. Here, we use a polarized hepatic cell line and in vivo models to demonstrate vectorial transport of HAV from hepatocytes into bile via the apical cell membrane. Although this transport is specific for HAV, the rate of fecal excretion in inefficient, accounting for less than 1% of input virus from the bloodstream per hour. However, we also found that the rate of HAV excretion was enhanced in the presence of HAV-specific IgA. Using mice lacking the polymeric IgA receptor (pIgR(-/-)), we show that a proportion of HAV:IgA complexes are transported via the pIgR demonstrating a role for specific antibody in pathogen excretion.

FcµR interacts and cooperates with the B cell receptor To promote B cell survival.

The IgM FcR (FcµR) promotes B cell survival, but the molecular mechanism remains largely unknown. We show using FcµR(-/-) and wild-type mice that FcµR specifically enhanced B cell survival induced by BCR cross-linking with F(ab')2-anti-IgM Abs while having no effect on survival when the B cells were activated by CD40 ligation or LPS stimulation. FcµR expression was markedly upregulated by anti-IgM stimulation, which may promote enhanced FcµR signaling in these cells. Immunofluorescence and confocal microscopy analyses demonstrated that FcµR colocalized with the BCR on the plasma membrane of primary B cells. Coimmunoprecipitation analysis further revealed that FcµR physically interacted with the BCR complex. Because NF-κB plays a prominent role in B cell survival, we analyzed whether FcµR was involved in BCR-triggered NF-κB activation. FcµR did not affect BCR-triggered IκBα phosphorylation characteristic of the canonical NF-κB activation pathway but promoted the production of the noncanonical NF-κB pathway component p52. Consistent with the elevated p52 levels, FcµR enhanced BCR-triggered expression of the antiapoptotic protein BCL-xL. Importantly, FcµR stimulation alone in the absence of BCR signaling had no effect on either IκBα phosphorylation or the expression of p52 and BCL-xL. Therefore, FcµR relied on the BCR signal to activate the noncanonical NF-κB pathway and enhance B cell survival. These results reveal a cross-talk downstream of FcµR and BCR signaling and provide mechanistic insight into FcµR-mediated enhancement of B cell survival after BCR stimulation.

Enhanced Pharmacokinetics of Factor VIIa as a Monomeric Fc Fusion.

Recombinant Factor VIIa (rFVIIa) is utilized for on-demand treatment of bleeding episodes in hemophilia patients with neutralizing antibodies (inhibitors) against Factor VIII or Factor IX, but a short half-life in the circulation (~2.5hrs) limits its use in a prophylactic setting. Recombinant FVIIa variants with improved pharmacokinetic properties may enable improved treatment and prevention of bleeding episodes in the inhibitor population. In this study we describe recombinant FVIIaFc (rFVIIaFc), a recombinant Fc-fusion protein generated to utilize the neonatal Fc receptor (FcRn)-mediated recycling pathway that protects immunoglobulin G from catabolism. On the basis of activity, rFVIIaFc exhibited a 5.5-fold extension in terminal half-life in hemophilia A mice compared to rFVIIa. The potency of rFVIIaFc was comparable to that of rFVIIa in thrombin generation assay and ROTEM. In agreement with these data, rFVIIaFc and rFVIIa showed similar acute efficacy at comparable molar doses in the tail clip bleeding model in hemophilia A mice. Taken together, these studies demonstrate enhanced pharmacokinetics and similar hemostatic properties for rFVIIaFc compared to rFVIIa.

ß2-Microglobulin deficiency causes a complex immunodeficiency of the innate and adaptive immune system.

BACKGROUND: Most patients with MHC class I (MHC-I) deficiency carry genetic defects in transporter associated with antigen processing 1 (TAP1) or TAP2. The clinical presentation can vary, and about half of the patients have severe skin disease. Previously, one report described ß2-microglobulin (ß2m) deficiency as another monogenetic cause of MHC-I deficiency, but no further immunologic evaluation was performed. OBJECTIVE: We sought to describe the molecular and immunologic features of ß2m deficiency in 2 Turkish siblings with new diagnoses. METHODS: Based on clinical and serologic findings, the genetic defect was detected by means of candidate gene analysis. The immunologic characterization comprises flow cytometry, ELISA, functional assays, and immunohistochemistry. RESULTS: Here we provide the first extensive clinical and immunologic description of ß2m deficiency in 2 siblings. The sister had recurrent respiratory tract infections and severe skin disease, whereas the brother was fairly asymptomatic but had bronchiectasis. Not only polymorphic MHC-I but also the related CD1a, CD1b, CD1c, and neonatal Fc receptor molecules were absent from the surfaces of ß2m-deficient cells. Absent neonatal Fc receptor surface expression led to low serum IgG and albumin levels in both siblings, whereas the heterozygous parents had normal results for all tested parameters except ß2m mRNA (B2M) expression. Similar to TAP deficiency in the absence of a regular CD8 T-cell compartment, CD8(+) γδ T cells were strongly expanded. Natural killer cells were normal in number but not "licensed to kill." CONCLUSION: The clinical presentation of patients with ß2m deficiency resembles that of patients with other forms of MHC-I deficiency, but because of the missing stabilizing effect of ß2m on other members of the MHC-I family, the immunologic defect is more extensive than in patients with TAP deficiency.

Increased serum IgA in Fcα/µR-deficient mice on the (129 x C57BL/6) F1 genetic background.

Fcα/µR (CD351) is an Fc receptor for both IgA and IgM, which is abundantly expressed in the small intestine. However, the role of Fcα/µR in the intestinal tissue is largely unknown. Here, we found that Fcα/µR is highly expressed on follicular dendritic cells (FDCs) in Peyer's patches (PP) in the small intestine. Fcα/µR-deficient mice on the (129 x C57BL/6) F1 background showed increased serum, but not fecal, IgA level in response to gut-oriented antigens. IgA(+) B cells were increased in PP, but not in the lamina propria, of Fcα/µR-deficient mice, which was attenuated after reduction of commensal microbiota by oral treatment with antibiotics. Analyses of bone marrow chimeric mice, in which either FDCs or blood cells or both lack the expression of Fcα/µR, suggested that FDCs, but not blood cells, were responsible for the increased serum IgA concentration in Fcα/µR-deficient mice. Moreover, Fcα/µR-deficient mice showed enhanced germinal center formation against commensal microbiota in PP. Thus, serum IgA production against gut-oriented antigens is negatively regulated by Fcα/µR on FDCs in the F1 mice.

Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection.

The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab')2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

Enhanced auto-antibody production and Mott cell formation in FcµR-deficient autoimmune mice.

The IgM-Fc receptor (FcµR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcµR(-) B6/lpr than FcµR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcµR(+) B6/lpr mice, were reduced to normal B6 levels in FcµR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcµR(-) B6/lpr mice compared with either FcµR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcµR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcµR(-) mice compared with FcµR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcµR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcµR(-) and FcµR(+) B6/lpr mice. Collectively, these findings suggest that FcµR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Fas (lpr/lpr) mice.

Abundant intracellular IgG in enterocytes and endoderm lacking FcRn.

FcRn, a non-classical MHCI molecule, transports IgG from mother to young and regulates the rate of IgG degradation throughout life. Brambell proposed a mechanism that unified these two functions, saying that IgG was pinocytosed nonspecifically by the cell into an FcRn-expressing endosome, where, at low pH, it bound to FcRn and was exocytosed. This theory was immediately challenged by claims that FcRn specificity for ligand could be conferred at the cell surface in neonatal jejunum. Assessing Brambell's hypothesis we found abundant nonspecifically endocytosed IgG present in the cytoplasm of FcRn(-/-) enterocytes. Further, IgG was present in the intercellular clefts and the cores of FcRn(+/+) but not FcRn(-/-) jejunum. FcRn specificity for ligand could be determined within the cell.

Mouse IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses.

FcR specific for pentameric IgM (FCMR) is expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity, and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.

Investigation of the role of FcγR and FcRn in mAb distribution to the brain.

To evaluate the role of Fc receptors (FcR) on IgG distribution to the brain, the disposition of 8C2, a murine monoclonal IgG1 antibody, was investigated after intravenous administration in FcRn α-chain knockout mice, FcγRIIb knockout mice, FcγRI/RIII knockout mice, and C57BL/6 control mice. (125)I-8C2 was co-administered with (51)Cr-labeled red blood cells to allow accurate assessment of residual blood content in brain samples. Blood and brain tissues were harvested from subgroups of three mice at several time-points up to 10 days, and radioactivity was counted. The blood and brain areas under 8C2 concentration vs time curves (AUCs) were calculated using the linear trapezoidal rule, and the associated standard deviations (SD) were assessed using a modified Bailer method. Concentration data were also analyzed with a semiphysiological population pharmacokinetic model. The brain/blood AUC ratios were comparable across all strains of mice (ratios ± SD): 0.00774 ± 0.000452, 0.00841 ± 0.000535, 0.00636 ± 0.000548, and 0.00917 ± 0.000478 for C57BL/6 control mice, FcγRI/RIII knockouts, FcγRIIb knockouts, and FcRn α-chain knockout mice (p > 0.05). Statistically significant improvement in model fitting of the data was shown with incorporation of a strain-specific parameter for antibody clearance for FcRn knockout mice; however, no significant improvements in model fitting were found for strain effects on any other parameter, including the brain uptake clearance or efflux clearances for 8C2. The predicted 8C2 brain efflux clearance was found to be ∼135-fold faster than the brain uptake clearance, consistent with the observed low ratio of brain-blood exposure. The experimental results and modeling results indicate that, in mice, FcRn and FcγR do not contribute to the "blood-brain barrier" that limits mAb uptake into the brain.

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses.

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM-antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcµR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcµR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcµR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcµR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcµR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcµR.