P. J. A. Capel

Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications

Receptors for the Fc domain of IgG (Fc gamma R) provide a critical link between specific humoral responses and the cellular branch of the immune system. When hFc gamma R interact with immunoglobulin, a variety of biological responses are triggered. These include phagocytosis, endocytosis, antibody-dependent cellular cytotoxicity (ADCC), release of inflammatory mediators, and enhancement of antigen presentation. In the last few years our understanding of the Fc gamma receptor structure has increased dramatically, due to the availability of monoclonal antibodies (mAb) and cDNA probes. Fc gamma R are members of the immunoglobulin superfamily and three main classes, hFc gamma RI, hFc gamma RII, and hFc gamma RIII are recognized in man generating at least 12 different isoforms. A further level of complexity is introduced by various genetic polymorphisms and, importantly, recent evidence points at the relevance of this Fc gamma R heterogeneity.

Impaired IgG-dependent anaphylaxis and Arthus reaction in FcγRIII (CD16) deficient mice.

Abstract The family of receptors for IgG (FcγR) plays an essential role in antibody-mediated effector functions of the immune system. However, the specific contribution of each of the FcγR classes to in vivo immune reactions is still unclear. Here, we demonstrate that mice deficient for the ligand-binding α chain of FcγRIII lack NK cell–mediated antibody-dependent cytotoxicity and phagocytosis of IgG1-coated particles by macrophages. Strikingly, these mice lack IgG-mediated mast cell degranulation, are resistant to IgG-dependent passive cutaneous anaphylaxis, and exhibit an impaired Arthus reaction. These results indicate a prominent role for FcγRIII in inflammatory and anaphylactic responses, making this receptor a potential target in immunotherapy.

On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets. Analysis of a functional polymorphism to human IgG2.

An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.

Antigen targeting to myeloid-specific human Fc gamma RI/CD64 triggers enhanced antibody responses in transgenic mice.

Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.

FUNCTIONAL ASSOCIATION BETWEEN THE HUMAN MYELOID IMMUNOGLOBULIN A FC RECEPTOR (CD89) AND FCR GAMMA CHAIN : MOLECULAR BASIS FOR CD89/FCR GAMMA CHAIN ASSOCIATION

FcR γ chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (FcεRI), and the class I and IIIA IgG receptors (FcγRI and FcγRIIIa). Here, we demonstrate that the Fc receptor γchain associates with FcαR in transfected IIA1.6 B lymphocytes. FcαR could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR γchain, a physical interaction with FcαR could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg209) within the transmembrane domain of FcαR promotes association with FcR γchain. We therefore constructed FcαR molecules where Arg209 was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between FcαR and FcR γchain was observed only with a positively charged residue (Arg209 or His209) present within the FcαR transmembrane domain. These data show that transmembrane signal transduction by the FcαR is mediated via FcR γchain, and that FcαR requires a positively charged residue within the transmembrane domain to promote functional association.

Chronic overproduction of islet amyloid polypeptide/amylin in transgenic mice : lysosomal localization of human islet amyloid polypeptide and lack of marked hyperglycaemia or hyperinsulinaemia

SummaryType 2 (non-insulin-dependent) diabetes mellitus is characterised by hyperglycaemia, peripheral insulin resistance, impaired insulin secretion and pancreatic islet amyloid formation. The major constituent of islet amyloid is islet amyloid polypeptide (amylin). Islet amyloid polypeptide is synthesized by islet beta cells and co-secreted with insulin. The ability of islet amyloid polypeptide to form amyloid fibrils is related to its species-specific amino acid sequence. Islet amyloid associated with diabetes is only found in man, monkeys, cats and racoons. Pharmacological doses of islet amyloid polypeptide have been shown to inhibit insulin secretion as well as insulin action on peripheral tissues (insulin resistance). To examine the role of islet amyloid polypeptide in the pathogenesis of Type 2 diabetes, we have generated transgenic mice with the gene encoding either human islet amyloid polypeptide (which can form amyloid) or rat islet amyloid polypeptide, under control of an insulin promoter. Transgenic islet amyloid polypeptide mRNA was detected in the pancreas in all transgenic mice. Plasma islet amyloid polypeptide levels were significantly elevated (up to 15-fold) in three out of five transgenic lines, but elevated glucose levels, hyperinsulinaemia and obesity were not observed. This suggests that insulin resistance is not induced by chronic hypersecretion of islet amyloid polypeptide. Islet amyloid polypeptide immunoreactivity was localized to beta-cell secretory granules in all mice. Islet amyloid polypeptide immunoreactivity in beta-cell lysosomes was seen only in mice with the human islet amyloid polypeptide gene, as in human beta cells, and might represent an initial step in intracellular formation of amyloid fibrils. These transgenic mice provide a unique model with which to examine the physiological function of islet amyloid polypeptide and to study intracellular and extracellular handling of human islet amyloid polypeptide in pancreatic islets.

Spontaneous liver tumors and Benzo[a]pyrene‐induced lymphomas in XPA‐deficient mice

Defects in the xeroderma pigmentosum complementation group A‐correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer‐prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet‐B and 7,12‐dimethylbenz[a]anthracene‐induced skin tumors. Here we report the XPA‐deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA‐deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P‐treated wild‐type and heterozygous mice. Our results show for the first time that XPA‐deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin. Mol. Carcinog. 19:46–53, 1997.

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B.

Xeroderma pigmentosum (XP) patients with a defect in the nucleotide excision repair gene XPA, develop tumors with a high frequency on sun-exposed areas of the skin. Here we describe that hairless XPA-deficient mice also develop skin tumors with a short latency time and a 100% prevalence after daily exposure to low doses of U.V.B. Surprisingly and in contrast to U.V.B.-exposed repair proficient hairless mice who mainly develop squamous cell carcinomas, the XPA-deficient mice developed papillomas with a high frequency (31%) at a U.V. dose of 32 J/m2 daily. At the highest daily dose of 80 J/m2 mainly squamous cell carcinomas (56%) and only 10% of papillomas were found in XPA-deficient hairless mice. p53 gene mutations were examined in exons 5, 7 and 8 and were detected in only 3 out of 37 of these skin tumors, whereas in tumors of control U.V.B.-irradiated wild type littermates this frequency was higher (45%) and more in line with our previous data. Strikingly, a high incidence of activating ras gene mutations were observed in U.V.B.-induced papillomas (in 11 out of 14 tumors analysed). In only two out of 14 squamous cell carcinomas we found similar ras gene mutations. The observed shift from squamous cell carcinomas in wild type hairless mice to papillomas in XPA-deficient hairless mice, and a corresponding shift in mutated cancer genes in these tumors, provide new clues on the pathogenesis of chemically- versus U.V.B.-induced skin carcinogenesis.

Characterization of Two Fc Receptors for Mouse Immunoglobulins on Human Monocytes and Cell Lines

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgG1 anti‐CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgG1 present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by a recently developed EA rosette assay, using the erythrocyte‐associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIgG1 is based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgG1 and does not bind mIgG2a or mIgG2b nor, surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG1 anti‐CD3‐induced T cell proliferation. mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR on human cell lines correlates well with their ability to support mIgG2a anti‐CD3‐induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specigic for mIgG2a (which also binds human IgG), and a second specific for mIgG1.

Synergism of interleukin 6 and 1 alpha,25-dihydroxyvitamin D3 in induction of myeloid differentiation of human leukemic cell lines.

Interleukin 6 (IL‐6) is a multifunctional cytokine with an important role in immunity. We analyzed the effect of recombinant human IL‐6 in combination with 1α,25‐dihydroxyvitamin D3 (Vit. D3) on differentiation of the human myeloid leukemic cell lines U937 and HL‐60 with respect to alterations in antigen expression and functional activity. Of a panel of antigens analyzed, only CD11b (the α chain of CR3), and GD14 (a cell surface protein recognizing the lipopolysaccharide‐binding protein‐lipopolysaccharide complex) had significantly increased expression. Expression of ICAM‐1 (CD54), a ligand for LFA‐1, was also found to be enhanced with a concomitant increase of ICAM‐1 mRNA levels. Enhanced nonspecific esterase levels and induction of respiratory burst activity confirmed that cell differentiation was induced. Furthermore, IL‐6 and Vit. D3 had a profound effect on functional activities, as shown by enhancement of rosetting between sheep erythrocytes, sensitized with C3bi (EAC), and either U937 or HL‐60 cells. Also, phorbol myristate acetate–induced homotypic adhesion of U937, which is ICAM‐1 dependent, was markedly induced by these agents. These results indicate an important role of IL‐6 and Vit. D3 in myeloid cell Function and development.