Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis.

INTRODUCTION: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1ß in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. METHODS: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1ß. RESULTS: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1ß was measured in mice treated with rFc-µTP-L309C. CONCLUSION: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1ß production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

IgG3 anti-Kell allotypic variation results in differential antigen binding and phagocytosis.

BACKGROUND: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT). STUDY DESIGN AND METHODS: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages. RESULTS: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients. CONCLUSION: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn.

Isoagglutinin-reduced immunoglobulin retains efficacy in mouse models of immune thrombocytopenia and rheumatoid arthritis and is less likely to cause intravenous immunoglobulin-associated hemolysis.

BACKGROUND: Immunoglobulin therapy including intravenous immunoglobulin (IVIg) has been used as an effective treatment for autoimmune/inflammatory conditions with few side effects. However, high-dose IVIg (1-2 g/kg) has been recognized as a cause of hemolytic anemia in non-blood group O patients. Hemolysis when observed has been due to anti-A/anti-B isoagglutinins contained in the IVIg. Recently, an isoagglutinin-reduced IVIg, whereby the anti-A and anti-B titers have been reduced by immunoaffinity chromatography, has been introduced; however, whether this new product is as efficacious as nonreduced immunoglobulin (Ig) or will result in less IVIg-associated hemolysis has not been resolved. STUDY DESIGN AND METHODS: We used in vitro phagocytosis by monocytes and proinflammatory/anti-inflammatory macrophages, with isoagglutinin-reduced and -nonreduced Ig opsonized group A1 , B, and A1 B red blood cells, to estimate clinical significance of the IgG isoagglutinins. We also used immune thrombocytopenia (ITP) and rheumatoid arthritis (RA) mouse models to examine the in vivo efficacy of isoagglutinin-reduced versus -nonreduced Ig on the amelioration of the diseases. RESULTS: In contrast to nonreduced Ig, phagocytosis was largely absent when isoagglutinin-reduced Ig was used at a concentration equivalent to a patient receiving 2 g/kg. The in vivo efficacy of isoagglutinin-reduced versus nonreduced Ig on the amelioration of experimental ITP and RA was similar, indicating no loss of efficacy due to the chromatographic removal of isoagglutinins. CONCLUSION: Isoagglutinin-reduced Ig should have efficacy similar to nonreduced Ig and result in less IVIg-associated hemolysis.

Using the K/BxN mouse model of endogenous, chronic, rheumatoid arthritis for the evaluation of potential immunoglobulin-based therapeutic agents, including IVIg and Fc-µTP-L309C, a recombinant IgG1 Fc hexamer.

BACKGROUND: High-dose intravenous immunoglobulin (IVIg), and more recently, subcutaneously-delivered Ig (SCIg), are used to treat a variety of autoimmune diseases; however, there are challenges associated with product production, availability, access and efficacy. These challenges have provided incentives to develop a human recombinant Fc as a more potent alternative to IVIg and SCIg for the treatment of autoimmune diseases. Recently, a recombinant human IgG1 Fc hexamer (Fc-µTP-L309C) was shown to be more efficacious than IVIg in a variety of autoimmune mouse models. We have now examined its efficacy compared to IVIg and SCIg in the K/BxN mouse model of endogenous, chronic rheumatoid arthritis (RA). RESULT: Using the serum-transfer K/BxN model and the endogenous autoimmune model, amelioration of the arthritis was achieved. Effective treatment required high and frequent doses of IVIg, SCIg and Fc-µTP-L309C. However, Fc-µTP-L309C was efficacious at 10-fold lower doses that IVIg/SCIg. Also, arthritis could be prevented when Fc-µTP-L309C was given prior to onset of the arthritis in both the endogenous model and in the serum transfer model. CONCLUSIONS: Our results show that Fc-µTP-L309C is a powerful treatment for the prevention and amelioration of severe, chronic arthritis in a true autoimmune mouse model of RA. Thus, the K/BxN endogenous arthritis model should be useful for testing potential therapeutics for RA. Our findings provide rationale for further examination of the treatment efficacy of immunoglobulin-based therapeutics in rheumatoid arthritis.

The Monocyte Monolayer Assay: Past, Present and Future.

Serologic testing using the indirect antiglobulin test (IAT) is known to be insufficient to determine the clinical significance or insignificance of a given antibody to red blood cells (RBC), particularly in cases of antibodies to high-prevalence antigens, such as anti-Ge or anti-Yta. An in vitro functional cellular assay, the monocyte monolayer assay (MMA), has been studied for more than 40 years for its potential use to differentiate between clinically significant and insignificant RBC antibodies. The MMA has recently been used to select donor blood for transfusion into patients having a serologically incompatible crossmatch, without any obvious sequalae. Thus, the MMA shows great potential for future use to select donor blood for transfusion in situations where antibodies to high-prevalence antigens or complex multiple alloantibody problems confront transfusion services. In this report, we review the history leading up to the current uses of the MMA, its optimization, and potential use for the selection of serologically incompatible donor blood for transfusion. We describe current ongoing work to document and improve the efficacy of the MMA. Finally, we briefly describe possible future directions to make the MMA more amenable to routine laboratories.

Differential regulation of IgA+ B cells in vitro by stromal cells from distinctive anatomical compartments.

B cell development is regulated by stromal cells (SCs) that form a supportive microenvironment. These SCs along with other cell types produce cytokines, chemokines, and adhesion molecules that guide B cell commitment and differentiation. BM, spleen (Sp), and the gut lamina propria (LP) constitute distinctive anatomical compartments that support B cell differentiation. In order to characterize and compare the signals necessary to generate IgA+ B cells, we developed an in vitro system to co-culture gut LP, BM, or Sp-derived SCs with B lineage cells. Using this co-culture system, we found that gut LP SCs promote IgA+ B cell accumulation through the production of soluble stimulatory factors. In contrast to gut LP SCs, BM and splenic SCs were found to impair IgA+ B cell accumulation in vitro. Taken together, these observations provide new insights into how SCs derived from different anatomical locations shape IgA+ B cell responses.

Bone marrow basophils provide survival signals to immature B cells in vitro but are dispensable in vivo.

Immature B cells are the first B cell progenitors to express a fully formed B cell receptor and are therefore subject to extensive selection processes that act to mitigate the emergence of autoreactive clones. While it is well appreciated that most B cell generation in the bone marrow is highly dependent on access to molecules present in the local milieu, the existence of extrinsically provided factors that modulate immature B cell biology is ambiguous. Nonetheless, a population of CD49b+CD90lo cells has demonstrated in vitro potential to promote immature B cell survival. Using a mouse basophil reporter strain we confirmed the identity of these CD49b+CD90lo supportive cells as basophils. However, analysis of bone marrow B cell populations following lineage specific basophil depletion demonstrates that basophils do not have a significant role in vivo in modulating immature B cell biology during steady-state conditions.

Inflammation rapidly reorganizes mouse bone marrow B cells and their environment in conjunction with early IgM responses.

Systemic inflammation perturbs the bone marrow environment by evicting resident B cells and favoring granulopoiesis over lymphopoiesis. Despite these conditions, a subset of marrow B cell remains to become activated and produce potent acute immunoglobulin M (IgM) responses. This discrepancy is currently unresolved and a complete characterization of early perturbations in the B-cell niche has not been undertaken. Here, we show that within a few hours of challenging mice with adjuvant or cecal puncture, B cells accumulate in the bone marrow redistributed away from sinusoid vessels. This response correlates with enhanced sensitivity to CXC chemokine ligand 12 (CXCL12) but not CXCL13 or CC chemokine ligand 21. Concurrently, a number of B-cell survival and differentiation factors are elevated to produce a transiently supportive milieu. Disrupting homing dynamics with a CXC chemokine receptor 4 inhibitor reduced the formation of IgM-secreting cells. These data highlight the rapidity with which peripheral inflammation modifies the marrow compartment, and demonstrate that such modifications regulate acute IgM production within this organ. Furthermore, our study indicates that conversion to a state of emergency granulopoiesis is temporally delayed, allowing B cells opportunity to respond to antigen.