J.G.J. van de Winkel

The Fc receptor γ-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor γ‐chain, the tyrosine kinase Syk and phospholipase Cγ2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor γ‐chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cγ2 by collagen stimulation is absent in mice lacking the Fc receptor γ‐chain. Tyrosine phosphorylation of phospholipase Cγ2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor γ‐chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein‐coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor γ‐chain or Syk. The absence of Fc receptor γ‐chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen‐ but not thrombin‐stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine‐based activation motif (ITAM) in signalling by a non‐immune receptor stimulus.

FcγRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection

The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

IgG receptor polymorphisms: risk factors for disease.

Abstract Two groups of receptors for immunoglobulin G (FcγR) can be distinguished. Endothelial cells and placental syncytiotrophoblasts express an MHC class I-like FcγR important for regulation of IgG half-life and IgG transport, respectively. FcγR expressed on leukocytes constitute a heterogeneous family of membrane bound and soluble proteins. The various FcγR (sub) classes of this family differ in ligand affinity and specificity, which is determined by primary structure, glycosylation, association with signaling subunits, and environmental factors (such as serine proteases). The finding that polymorphisms of FcγRIIa, FcγRIIIa, and FcγRIIIb critically affect interaction with antibodies has prompted analysis in patients which provided tantalizing evidence for the relevance of FcγR polymorphisms as risk factors for a number of infectious and autoimmune diseases. Two groups of receptors for immunoglobulin G (FcγR) can be distinguished. Endothelial cells and placental syncytiotrophoblasts express an MHC class I-like FcγR important for regulation of IgG half-life and IgG transport, respectively. FcγR expressed on leukocytes constitute a heterogeneous family of membrane bound and soluble proteins. The various FcγR (sub) classes of this family differ in ligand affinity and specificity, which is determined by primary structure, glycosylation, association with signaling subunits, and environmental factors (such as serine proteases). The finding that polymorphisms of FcγRIIa, FcγRIIIa, and FcγRIIIb critically affect interaction with antibodies has prompted analysis in patients which provided tantalizing evidence for the relevance of FcγR polymorphisms as risk factors for a number of infectious and autoimmune diseases.

Relevance of Fcγ Receptor and Interleukin-10 Polymorphisms for Meningococcal Disease

The contribution of individual Fcgamma receptor (FcgammaR) subclasses to meningococcal phagocytosis was studied. In addition, functional FcgammaR polymorphisms were determined in 50 patients with meningococcal disease (MD), in 183 first-degree relatives of MD patients, and in 239 healthy control subjects, to study the association of FcgammaR genotypes with disease. Efficient internalization of opsonized Neisseria meningitidis serogroup B was mediated via multiple FcgammaR subclasses on phagocytes. Accordingly, a low-efficiency combination of FcgammaRIIa-R/R131, FcgammaRIIIa-F/F158, and FcgammaRIIIb-NA2/2 genotypes was increased significantly in relatives of patients with MD, compared with healthy control subjects (P<.05; odds ratio, 2.6; 95% confidence interval, 1.1-6.3). FcgammaRIIa and FcgammaRIIIa genotype distributions differed between patients with sepsis and those with meningitis. Combined genotypes of FcgammaRIIa and interleukin-10 -1082, which was previously reported as being associated with MD outcome, were distributed randomly in control subjects but not in relatives of patients with MD (P<.01). These data provide further evidence for the association of polymorphic genes on chromosome 1 and MD.

Role of Fc receptor γ chain in inflammation and cartilage damage during experimental antigen‐induced arthritis

OBJECTIVE To study the role of Fc receptor (FcR) gamma chain in inflammation and cartilage destruction during antigen-induced arthritis (AIA). METHODS FcR gamma-/- mice and controls were immunized with methylated bovine serum albumin (mBSA) in Freunds complete adjuvant, followed by induction of arthritis by local injection of mBSA into the right knee joint. Joint inflammation was studied by 99mTc uptake and by histology. Breakdown of proteoglycans from the cartilage matrix was determined by loss of red staining in Safranin O-stained knee joint sections, and matrix metalloproteinase (MMP)-mediated aggrecan degradation was determined by immunolocalization using anti-VDIPEN antibodies. Chondrocyte death was measured by determining empty lacunae in hematoxylin-stained sections and with the TUNEL assay in cryostat sections. Erosion was detected as ruffling of the cartilage surface. RESULTS Joint swelling, as measured by 99mTc uptake on days 1, 3, and 7, was significantly decreased in FcR gamma-/- mice compared with controls. On day 7 after AIA induction, sustained joint inflammation, as seen histologically, was not significantly lower in FcR gamma-/- deficient mice. In various cartilage layers (femur, tibia, patella) of central arthritic knee joints, marked depletion of proteoglycans (40-70%), chondrocyte death (25-50%), and mild surface erosion were found. In FcR gamma-/- knee joints, depletion of proteoglycans was comparable (40-70%). Strikingly, chondrocyte death and matrix erosion were absent. Furthermore, MMP-induced aggrecan neoepitopes, which were abundantly found in controls, were also absent in FcR gamma-/-. Nevertheless, latent MMPs were present in the cartilage matrix as seen in APMA-activated patellae. CONCLUSION FcR gamma chain is involved in the severity of acute and sustained inflammation and is a crucial factor in cartilage erosion during AIA, probably by regulating activation of latent MMPs present in the cartilage matrix.

A novel PCR-based method for direct Fcγ receptor IIIa (CD16) allotyping

Abstract Leukocyte IgG receptors (FcγR) are important immune-response modulating molecules. FcγRIIIa is expressed on macrophages, NK-cells and γδ-T cells and exhibits a genetically determined, functional polymorphism at nucleotide 559. This allelic difference predicts either a phenylalanine (F158) or valine (V158) at amino acid 158 in the membrane-proximal extracellular domain, and has been shown to be associated with autoimmune and infectious diseases. Published methods to determine FcγRIIIa genotypes are cumbersome. Therefore, we developed a novel, rapid and reliable PCR-based method to determine FcγRIIIa genotypes. Comparison of genotyping results with direct FcγRIIIa sequencing of 60 blood donors showed 100% accuracy of this new method. Since genotype frequencies of FcγR polymorphisms depend strongly on race and ethnicity, we compared FcγRIIIa genotype frequencies of 176 Caucasian Dutch and 104 Japanese blood donors. Interestingly, these frequencies were not significantly different (P>0.1), in contrast to the FcγRIIa and FcγRIIIb genotype frequencies (P

Phase I clinical trial of the bispecific antibody MDX-H210 (anti-FcγRI x anti-HER-2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer

A phase I study of the bispecific antibody MDX-H210 in combination with granulocyte colony-stimulating factor (G-CSF) was performed in stage IV breast carcinoma patients, overexpressing HER-2/neu. MDX-H210, constructed by crosslinking antigen binding fragments (F(ab′) fragments) of monoclonal antibody (mAb) H22 to Fc gamma receptor I (FcγRI), and mAb 520C9 to HER-2/neu, respectively, mediates the lysis of tumour cells in vitro, and in human FcγRI transgenic mouse models. The proto-oncogene HER-2/neu is overexpressed in approximately 30% of breast cancer patients, and represents a promising target for antibody-based immunotherapy. Fc gamma receptor I (CD64) is an effective trigger molecule, which is expressed on monocytes/macrophages, immature dendritic cells, and G-CSF-primed polymorphonuclear cells (PMN). Patients received G-CSF (Filgrastim) for 8 consecutive days, and cohorts of three patients were treated on day 4 with escalating, single doses of MDX-H210. A total of 30 patients were included, and treatment was generally well tolerated, without reaching dose-limiting toxicity. Side effects consisted mainly of fever and short periods of chills, which were timely related to elevated plasma levels of interleukin 6 and tumour necrosis factor alpha. In the last two cohorts, MDX-H210 plasma levels exceeded 1 μg ml−1, and on circulating myeloid cells >50% saturation of FcγRI was found until day 4. These effector cells were highly effective in antibody-dependent cell-mediated cytotoxicity. Immunohistochemical analyses of tumour biopsies in individual patients documented infiltration of monocytes and PMN after MDX-H210 infusion. Although the clinical course of the disease was not altered by the single dose of MDX-H210, a favourable toxicity profile – even at high doses – and remarkable biological effects were seen when combined with G-CSF. Therefore, the combination of G-CSF and MDX-H210 should be evaluated in further immunotherapeutical strategies.

IgG receptor IIa alleles determine susceptibility and severity of Guillain-Barré syndrome

Objective: Guillain-Barré syndrome (GBS) is characterized by nerve infiltration of leukocytes and autoantibodies of the immunoglobulin G (IgG) isotype directed against nerve constituents. Leukocyte receptors for IgG (FcγR) constitute an important link between the humoral and cellular parts of the immune system and confer potent cellular effector functions to myelin-directed antibodies. Three FcγR subclasses exhibit genetically determined biallelic functional polymorphisms (FcγRIIa: R131 versus H131; FcγRIIIa: 158V versus 158F; FcγRIIIb: NA1 versus NA2) that determine efficacy of the cellular immune response. To study the relevance of these polymorphisms for susceptibility and severity of GBS, we compared FcγR genotype distributions in GBS patients with those in controls. Methods: Genomic DNA was isolated from whole blood of 31 randomly selected patients with GBS and 187 healthy blood donors. Genotypes of the three polymorphic FcγR genes were determined by PCR. Results: FcγRIIa-H131 homozygosity was significantly increased in patients as compared with healthy controls (OR 2.45; 95% CI 1.12 to 5.36; p = 0.037). Furthermore, FcγRIIa-H131 homozygous GBS patients had a higher risk for severe disease than did patients with other genotypes (OR 18.57; 95% CI 1.95 to 176.49; p = 0.007). Conclusion: FcγRIIa allotypes capable of initiating efficient cellular effector functions are associated with increased risk for GBS and a more severe disease course. FcγR alleles may constitute novel genetic risk markers for GBS.

Expression of Fc receptors for IgG during acute and chronic cutaneous inflammation in atopic dermatitis

Atopic dermatitis is an allergic skin disease characterized by elevated total and antigen‐specific serum IgE and IgG4 levels. In acute and chronic cutaneous inflammation, large cellular infiltrates including T cells, dendritic cells and macrophages are found, especially in the dermis. These cells play an important part in the regulation of local inflammatory reactions. Receptors binding IgG (FcγR) are involved in dendritic cell and macrophage function. In this study, we examined the in vivo distribution and cellular expression of the three classes of leucocyte FcγR in human skin during acute and chronic cutaneous inflammation in atopic dermatitis. Atopy patch test skin was used as a model for acute inflammation in atopic dermatitis, while chronic lesional skin was used to investigate FcγR expression in chronically inflamed skin. In atopy patch test sites no increase in the number of CD1a+ dendritic cells and a slight increase in macrophages compared with non‐lesional skin was observed. Our results showed increased expression of FcγRI (CD64) and FcγRIII (CD16) in acutely inflamed skin as well as in chronically inflamed lesional skin, compared with healthy and non‐lesional atopic dermatitis skin. FcγRI was expressed by RFD1+, RFD7+ and CD68+, but not by CD1a+ dermal dendritic cells. RFD1+ dendritic cells and CD68+ macrophages were the main FcγRIII‐expressing cells during the acute inflammatory reaction. The significant increase in expression of FcγRIII (CD16) and FcγRI (CD64) probably results from upregulation of the receptors on resident cells. Insight into the presence of FcγR+ cells in human skin during inflammation is important both for our understanding of skin immune reactions and the development of new therapeutic concepts.

In situ depletion of CD4+ T cells in human skin by Zanolimumab

CD4+ T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4+ T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, κ) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4+, but not CD8+ CD3+ T cells. The capacity of Zanolimumab to deplete the CD4+ T cells in the skin may be of importance in diseases where CD4+ T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.