Clearance of red cells by monoclonal IgG3 anti-D in vivo is affected by the VF polymorphism of Fcgamma RIIIa (CD16)

Human red cells (RBC) coated with IgG anti-D are cleared from the circulation to the spleen by macrophages which express IgG receptors (Fcgamma R). Polymorphisms of Fcgamma RIIa and Fcgamma RIIIa affect IgG binding in vitro, and may alter the efficiency of clearance of immune complexes in vivo. In a RBC clearance study, 22 Rh D-negative subjects were given 100-400 micro g human monoclonal or polyclonal IgG anti-D i.m. followed 48 h later by 51Cr-labelled D+ RBC. The half lives of the infused D+ RBC were determined, together with the coating levels of anti-D on the D+ RBC. Fcgamma RIIA and FcgammaRIIIA genotyping was performed. Large ranges of phagocytosis and extracellular lysis of RBC in vitro, and of half lives of RBC in vivo, were observed. Clearance of RBC coated with monoclonal IgG3 anti-D (BRAD-3) was more rapid in five subjects homozygous for Fcgamma RIIIa-F/F158 than in three subjects expressing the Fcgamma RIIIa-V158 allele (P = 0.024). This effect was not observed, however, for those individuals given polyclonal anti-D. There was also no significant difference in the efficiency of RBC destruction in vitro or of RBC clearance in vivo between the subjects analysed for individual genotypes or alleles or combinations of alleles. In conclusion, the presence of the Fcgamma RIIIa-V158 allele compromised the efficiency of removal of RBC coated with IgG3 anti-D.     

Genomic organization of classical human low-affinity Fcgamma receptor genes

The classical low-affinity Fcgamma receptor genes (FcgammaRIIA, B, C and FcgammaRIIIA, B) are located on chromosome 1q23, a region that shows strong linkage with human systemic lupus erythematosus (SLE) in several genome-wide scans, and family-based association between FcgammaRIIIA and SLE is now established. High homology among the Fcgamma receptor genes, however, has hampered further study of this region. We have used a human bacterial artificial chromosome (BAC) library to determine the order and orientation of these Fcgamma receptor genes and have sequenced the very highly homologous 5' region (including 3.4 kb of the promoter and the 8 kb from exon 1 to exon 3) of the FcgammaRIIB and FcgammaRIIC genes to enable study of their unique single nucleotide polymorphisms (SNP). We have utilized these data to characterize a linked set of three coding region SNPs in the FcgammaRIIC exon 3 (EC1) that includes the stop codon SNP, which provides an important insight into natural killer cell function. Together, these data provide the basis for the study of additional SNPs in FcgammaR genes in SLE disease susceptibility.     

Evaluation of aggregated IgG in mice as an Fc receptor specific probe of the hepatic mononuclear phagocyte system.

In experimental models of immune complex diseases the hepatic mononuclear phagocyte system removes circulating immune complexes (CIC) by interaction with Fc receptors, and the spleen has a relatively insignificant role in this function. We have used heat-aggregated human IgG (AHGG) to detect altered hepatic mononuclear phagocyte system activity in an acute immune complex model in mice in order to evaluate its suitability for possible use in humans. Immune complexes inhibited the clearance of AHGG, as a function of the dose and of the time after injection of complexes. The delayed clearance resulted from decreased hepatic uptake of the AHGG. Alterations in the comparatively small splenic uptake of AHGG did not correlate with changes in the clearance or the hepatic uptake that were produced by the complexes. Studies with Rose Bengal showed that the complexes caused a small but definite decrease in hepatic blood flow. Immune complexes also inhibited the clearance and hepatic uptake of aggregated mouse albumin and aggregated ovalbumin. The aggregated albumins, however, were cleared very rapidly, indicating high extraction ratios, so their clearance was more affected by the decreased blood flow than the clearance of AHGG. We conclude that a small dose of AHGG is a sensitive probe for hepatic Fc receptor function and has potential for human use.     

Macrophage Fcgamma receptors expression is altered by treatment with dopaminergic drugs

Macrophage Fcgamma receptors have an important role in host defense and the pathophysiology of immune mediated disorders. Alteration of splenic macrophage Fcgamma receptors expression predisposes to severe infection. Inhibition or blockade of splenic macrophage Fcgamma receptors is one of the mechanisms by which immune cytopenias improve. Dopaminergic drugs have clinically significant regulatory functions on the immune response. Using an experimental model in the guinea pig we assessed the effect of commonly used dopaminergic drugs on the expression of macrophage Fcgamma receptors. Three dopa-antagonists, bromocryptine, leuprolide, and pergolide, and seven dopa-antagonists, chlorpromazine, SCH 23390, metochlopramide, sulpiride, veralipride, alizapride, and cisapride, were studied. Following guinea pig treatment with dopaminergic drugs, the clearance of IgG-sensitized RBCs in vivo, the in vitro binding of IgG-sensitized RBCs by isolated splenic macrophages and flow cytometry with monoclonal antibodies were performed. Treatment with dopa-agonists enhanced the clearance of IgG-sensitized RBCs, the in vitro binding of IgG-sensitized RBCs by isolated splenic macrophages, and the cell surface expression of both macrophage Fcgamma receptors, and vice versa, dopa-antagonists impaired macrophage Fcgamma receptors expression. Macrophage FcgammaR1,2 was more sensitive than FcgammaR2 to such dopaminergic effect. These alterations of macrophage Fcgamma receptors expression are mediated by both D1 and D2 dopamine receptors, with a major participation of D2 receptors. Dopaminergic drugs alter the clearance of IgG-coated cells by an effect at the expression of splenic macrophage Fcgamma receptors.     

Human neutrophil Fcgamma receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases

Inflammation mediated by antibody-antigen complexes contributes to autoimmune diseases. Mice deficient in the common Fcgamma-chain are protected from IgG-mediated glomerulonephritis and the reverse passive Arthus (RPA) reaction and FcR-bearing macrophages, and mast cells have been assigned primary roles in these processes. Here we demonstrate that neutrophil-selective transgenic expression of the two uniquely human neutrophil Fc gamma receptors (FcgammaRs), FcgammaRIIA and FcgammaRIIIB, in Fcgamma-chain-deficient mice restored susceptibility to progressive glomerulonephritis and the cutaneous RPA reaction. FcgammaRIIIB and FcgammaRIIA mediated neutrophil accumulation, whereas FcgammaRIIA alone promoted organ injury. In a model of soluble immune complexes deposited within the vasculature, FcgammaRIIIB was responsible for neutrophil slow rolling and adhesion whereas in the cremaster RPA, induced by both vascular and tissue soluble immune complexes, FcgammaRIIA predominated. Thus, human FcgammaRs on neutrophils serve as molecular links between antibody and immunological disease, with FcgammaRIIA promoting tissue injury and FcgammaRIIIB and FcgammaRIIA displaying specialized context-dependent functions in neutrophil recruitment.     

Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization

Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.     

Experimental epithelioid cell granulomas, tubercle formation and immunological competence: an ultrastructural analysis

An ultrastructural study of the cells comprising tubercles in experimental mycobacterial granulomas in rats is presented. Tubercle formation was compared in: (1) primary infections due to BCG at 49 days, (2) reinfection with BCG at 7 days, 8 months after primary infection and (3) lesions due to preformed BCG/anti-BCG complexes in antibody excess in unprimed animals at 10 days. The most rapid elimination of antigen with resolution of the lesion in the reinfection lesions, was effected by the early recruitment of cells morphologically characteristic of activated macrophages and immature epithelioid cells. The next best performance was in the immune complex lesion which at its height was maintained by a roughly constant size due to promonocytes, monocytes, macrophages, activated macrophages and immature epithelioid cells accumulated at the site. True epithelioid cells were rare. The slowest rate of healing in the primary 49 day granulomas, was associated with the whole spectrum of mononuclear phagocyte series of cells. All granulomas were surrounded by a cuff of mixed lymphocytes, interdigitating cells and fibroblasts. The rapid resolution of the reinfection lesions appeared as a loosely compacted infiltration of cells which allowed intimate contact of lymphocytes and macrophages. This was distinct from the tightly compared and compartmentalised zones of lymphocytes and macrophages which inhibited contact in the most slowly resolving lesions. An intermediate arrangement was seen in the immune complex granuloma. Another factor which predisposed to rapid resolution was the high vascularity of the central region of the granulomas. In the slow resolving group vascularity was diminished and peripherally situated.     

Differentiation of the human monocyte cell line,U937, with dibutyryl cyclicAMP induces the expression of the inhibitory Fc receptor,FcgammaRIIb

FC receptor for IgG receptor (Fcgamma) mediated activation of macrophages is essential for the clearance of immune complexes and control of inflammation. However, activated macrophages play an integral role in tissue destruction associated with autoimmune and inflammatory disease processes. Understanding the mechanisms which balance activating and inhibitory signals generated by immune complexes are therefore of critical importance to human disease. Here, we demonstrate that differentiation of the human monocytic U937 cell line to a macrophage phenotype with dibutyryl cyclicAMP induces both mRNA and protein expression of the inhibitory IgG receptor, FcgammaRIIb1. We further demonstrate that, following receptor aggregation, FcgammaRII transiently recruits the 5'-inositol phosphatase, SHIP. These data define a role for FcgammaRIIb in the modulation of immune complex mediated macrophage activation in a human model system.     

The Placental Fcγ Receptors Studied Using Immune Complexes of Peroxidase

Immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP were used to study the Fcgamma receptors in normal human placenta. Cryostat sections of placental tissue were incubated with the complexes, and the peroxidase activity was revealed histochemically. The bound complexes were localized to the apical surface of the trophoblasts and endothelial cells of the fetal stem vessels. Binding also occurred within the wall of some fetal vessels, to stromal cells and occasionally to areas corresponding to the trophoblastic membrane. The strongest binding was obtained with immune complexes prepared at slight antigen excess. Eight- to sixteen-fold increased concentration of human and rabbit IgG was needed to block the binding of immune complexes. Bovine and porcine IgG did not block the binding. Treatment of tissue sections with neuraminidase enhanced the binding activity of the receptors. The technique is very convenient for studies of Fcgamma receptors in tissue. However, unfixed frozen placental tissue was not suitable for ultrastructural studies.     

FcgammaRIII is protective against Pseudomonas aeruginosa pneumonia

Defenses against bacterial infections involve activation of multiple systems of innate immunity, including complement, Toll-like receptors, and defensins. Reactions to chronic infections bring adaptive immune mechanisms into play as well, with the introduction of modulatory interactions between the two. In humans with chronic lung infections, the severity of inflammation and disease correlate with elevated levels of pathogen-specific immune complexes and complement activation. In mice with genetic deficiency in C5, or targeted deletion of the C5a receptor, Pseudomonas lung infections reveal a role for the C5a anaphylatoxin in disease severity. Deficient animals exhibit significantly reduced survival and clearance of infecting bacteria, simultaneous with greatly increased pulmonary influx of inflammatory cells. Among the actions of C5a on inflammatory cells mediated through the C5a receptor is a shift in the relative expression of Fcgamma receptors to increase FcgammaRIII relative to FcgammaRII. This shift may significantly impact defenses against chronic infection, reflecting the cellular activation profiles of these IgG receptors. We addressed the role of FcgammaRIII in defense against Pseudomonas lung infection, and found that, like C5aR-deficient mice, animals with targeted deletion of FcgammaRIII are more susceptible to mortality upon infection and exhibit reduced clearance of the pathogen. Pseudomonas infection was associated with an increase in the FcgammaRIII/FcgammaRII ratio in wild-type mice, and the data support its role as an additional mechanism of host defense against bacterial infection.     

Both Fcgamma and complement receptors mediate transfer of immune complexes from erythrocytes to human macrophages under physiological flow conditions in vitro

Abnormal clearance by the mononuclear phagocytic system of immune complexes (IC) is important in the pathogenesis of systemic lupus erythematosus (SLE). We have developed an in vitro model to investigate the cellular mechanisms involved in the transfer of soluble IC from erythrocytes to human macrophages under physiological flow conditions. In this assay, erythrocytes bearing fluorescently labelled IC are perfused over monolayers of human monocytes or monocyte-derived macrophages in a parallel-plate flow chamber, and transfer quantified using confocal microscopy and flow cytometry. Using aggregated human IgG as a model IC, we have been able to demonstrate transfer of IC from erythrocytes to macrophages. Blocking studies with specific neutralizing antibodies have shown that both complement and Fcgamma receptors are required for IC transfer. Blockade of CR4 (alpha(x)beta(2) integrin), FcgammaRIIa or FcgammaRIII reduced transfer, while anti-CR3 (alpha(m)beta(2) integrin) had no effect. Blockade of CR3, FcgammaRIIa or FcgammaRIII also reduced the number of adhesive interactions between fluorescently labelled IC-bearing erythrocytes and macrophage monolayers. Taken together with the transfer data, this suggests differing roles for these receptors in the human IC transfer reaction that includes an adhesive function which facilitates IC processing by mononuclear phagocytes. Finally, a functional effect of the FcgammaRIIa R131/H131 polymorphism, important in susceptibility to SLE, has also been demonstrated using this model. Uptake of IgG(2) but not IgG(1)-containing soluble IC was reduced by macrophages from individuals homozygous for the R131 allelic variant of the receptor.     

Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages.

A variable population of fat-filled "foam" cells in diet-induced experimental arterial intimal plaques of rabbits and monkeys were analyzed for several features characteristic of macrophages. These included: 1) surface binding and phagocytosis of antibody-coated or complement-coated erythrocytes to detect specific surface receptors; 2) cytochemical tests and ultrastructural features to evaluate cell function and structure; and 3) rapid adherence to glass, a feature of macrophage activity, to isolate and identify a homogeneous population of fat-filled foam cells from excised and disrupted arterial lesions. Mixed populations of cells grown in culture from explants of lesions were also analyzed and lipid-filled cells were studied in histologic sections of adjacent lesions. Eighty to ninety percent of the easily dislodged glass-adherent cells from lesions had surface receptors for the Fc portion of immunoglobulin G and for the third component of complement. Coated red blood cells were readily phagocytized, but noncoated cells were not. Acid lipase activity was demonstrated in the Fc-receptor-positive cells. These cells were also devoid of ultrastructural features of smooth muscle. Among the cells growing or migrating out of explants, a population of large round foam cells possessed all of the macrophage features found in the glass-adherent cells from lesions and lacked ultrastructural characteristics of smooth muscle. Fusiform lipid vacuolated cells also grew out of the explants but did not exhibit surface receptors, failed to phagocytize coated or noncoated erythrocytes and did not stain for acid lipase activity; these cells showed distinctive morphologic features of smooth muscle. In histologic sections of nearby lesions foam cells that showed macrophage characteristics, ie, acid lipase activity and the presence of lysozymelike antigen, lacked ultrastructural smooth muscle features. Smooth muscle cells in lesion sections often contained lipid but demonstrated no lysozyme or acid lipase activity. The occurrence of a population of cells with several functional and structural features of macrophages among the lipid-laden cells of experimental diet-induced arterial lesions suggests that some foam cells may be derived from monocytes. An alternative explanation, that metabolically altered autochthonous arterial wall cells assume one or more characteristics of mononuclear phagocytes is less likely, since some of the markers used in these experiments are unrelated. Both explanations deserve further careful study.     

Fc gamma R expression on NK cells influences disease severity in rheumatoid arthritis

Rheumatoid arthritis (RA) is associated with autoantibodies, the best known of which is rheumatoid factor (RF). RF/IgG complexes interact with FcgammaR on the surface of neutrophils, NK cells and monocyte/macrophages. We have analyzed the expression pattern and allelic polymorphisms of three FcgammaR genes (FcgammaRIIA, FcgammaRIIC and FcgammaRIIIA) in a large sample of RA patients and normal donors. We have found that the level of FcgammaR (CD16 and CD32) expression on NK cells is lower in RA patients than in normal individuals. Genotypic analysis demonstrated that the CD32 isoform expressed by the majority of RA patients was not the activating FcgammaRIIc1 isoform, commonly seen in normal individuals, but rather the inhibitory FcgammaRIIb isoform. The combination of the FcgammaRIIIA-176F allele with a lack of CD32 expression in NK cells appeared to be characteristic of RA subjects with aggressive disease. Since FcgammaRII and FcgammaRIIIA are predominantly expressed by NK cells, these data further suggest that FcgammaR-mediated activation of NK cells could be a disease-determining factor in RA patients.     

Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcgammaRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcgammaR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src-related tyrosine kinases (SRTKs) and phosphatidyl inositol 3-kinases (PI3K) play important roles in FcgammaR phagocytosis. Herein, we demonstrate that these kinases are not required for FcgammaR endocytosis. Endocytosis of heat-aggregated IgG (HA-IgG) by COS-1 cells stably transfected with FcgammaRIIA or chimeric FcgammaRI-gamma-gamma (EC-TM-CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG-sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow-derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcgammaRIIA was cross-linked using HA-IgG or EA. However, inhibition of FcgammaR phosphorylation prevented only FcgammaR phagocytosis. Finally, biochemical analyses of PI3K(p85)-Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcgammaR phagocytosis and endocytosis. Overall, our results indicate that FcgammaR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition-based model in which PI3K and c-Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.     

Modulation of mononuclear phagocyte system function and circulating immune complexes by lyophilized concentrates in patients with classic hemophilia

Infusions of lyophilized antihemophilic factor concentrates in patients with hemophilia were found to affect both circulating immune complexes and in vivo mononuclear phagocyte system function. Following infusion nine patients had simultaneous assessment of serial immune complex levels and mononuclear phagocyte system clearance of IgG-sensitized autologous erythrocytes relative to a previously established baseline. Nine patients also had a separate second sequence, and two a third sequence, of serial immune complex measurements in relation to infusions. The net change in immune complexes over the 2- to 4-hr interval following infusion was consistent in 10 of 11 study pairs (P less than 0.01) despite different antihemophilic factor preparations for each study and different individual patient responses. This change could not be explained by immunochemical rearrangement in infusate and serum since in vitro mixing experiments showed no relationship to in vivo results. Change in mononuclear phagocyte system function showed a strong correlation with change in complexes (r = 0.70; P less than 0.05). It is suggested that infusions of antihemophilic factor can modulate the mononuclear phagocyte system which in turn alters immune complex levels. Both of these effects could potentially influence immune regulation which has been shown to be abnormal in other hemophiliac patient groups.     

Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells.

Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo.     

Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells

Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo.     

Class II major histocompatibility antigen expression on coronary arterial endothelium in a patient with Kawasaki disease

To investigate the class II major histocompatibility antigen expression on coronary arterial endothelium of Kawasaki disease and immunophenotypes of the infiltrating cells in the coronary vascular lesions, myocardial sections from a patient who died during the acute stage of Kawasaki disease were studied using an immunoperoxidase technique. The mononuclear cells in the lesions mainly consisted of macrophages and T cells, whereas B cells and NK/K cells were not seen. The majority of T cells reacted with Leu-3a antibodies, and only a few reacted with Leu-2a antibodies. Cells bearing the interleukin-2 receptor, indicative of activated T cells, were also found in the lesions. To determine the distribution of class II antigen, we used anti-HLA-DR antibodies. The massive expression of HLA-DR antigen on mononuclear cells was found in the lesions. In addition, the HLA-DR activation antigen was expressed on the coronary arterial endothelium at the infiltrates in which macrophages and T cells coexisted. In contrast, coronary arterial endothelium did not express HLA-DR antigens in the myocardial tissues of controls (n = 4). HLA-DR+ endothelial cells may play an important role in the development of Kawasaki vasculitis.     

Macrophage interactions with antibodies and soluble immune complexes

In vitro studies aimed at characterising (1) the binding of monomeric immunoglobulins from a variety of animal species to homologous mononuclear phagocytes, (2) the enhancement in phagocyte binding when antibodies are reacted with soluble antigens to form complexes of defined size, (3) the kinetics of complex ingestion and catabolism by macrophages and the biochemical mechanisms involved, (4) the role of complement in soluble complex catabolism and (5) the stimulatory effects of soluble complexes on phagocyte activity are reviewed. Insights gained from these studies into the in vivo clearance of soluble complexes and into the part played by circulating immune complexes in disease are discussed.     

The presence of IgE on macrophages and dendritic cells infiltrating into the skin lesion of atopic dermatitis

Monoclonal antibodies reactive with human IgE were used to investigate the presence of surface IgE in situ on mononuclear cells infiltrating into the skin lesion of atopic dermatitis (A.D.) by application of the immunoperoxidase technique to tissue sections for light and electron microscopic examination. A substantial proportion of infiltrating macrophages but not of lymphocytes were found to bear IgE on their cell surfaces. These observations raise the possibility that IgE may contribute to the skin inflammation associated with A.D. via non-mast-cell-mediated immune mechanisms. We hypothesize that allergens introduced into the skin lesion of A.D. are potentially capable of interacting not only with IgE-bearing mast cells but also with IgE-bearing macrophages and dendritic cells to cause the release of inflammatory mediators.