IgG-sensitized Erythrocytes Research Articles

Gammaglobulin for intravenous use induces an Fc γ receptor-specific decrement in phagocytosis by blood monocytes

Intravenous gammaglobulin (IVIG) increases the circulating platelet count in patients with idiopathic thrombocytopenic purpura in association with mononuclear phagocyte system blockade and decreased blood monocyte phagocytosis. To investigate whether IVIG treatment induces an Fc receptor-specific defect or a generalized phagocytic dysfunction which might impair host defenses, we studied the influence of IVIG on Fc receptor-dependent and Fc receptor-independent internalization by monocytes. In purified monocytes incubated in suspension with IVIG (10 mg IgG/ml) for 48 hr, our data demonstrate that Fc receptor-mediated phagocytosis of IgG-sensitized erythrocytes (EA) is significantly decreased. By contrast, there was no significant change in the phagocytosis of neuraminidase-treated erythrocytes which are internalized via the B-glucan receptor. A similar decrease in EA ingestion was observed in adherent monocytes incubated with IVIG. By contrast, the capacity of IVIG-treated adherent monocytes to internalize tannic acid-treated erythrocytes, an Fc receptor-independent probe, was the same as that of control cells. These findings demonstrate that IVIG does not induce a generalized phagocytic blockade, but rather a selective deficit in Fc γ receptor-mediated internalization. Thus, Fc receptor-independent mechanisms may provide adequate mononuclear phagocyte system function for host defense in patients receiving IVIG.

Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.

Bone marrow cells (BMC) flushed from femora of Lewis rats were cultured in Dulbecco's modification of Eagle's medium supplemented with mouse L929 cell supernatant as a source of colony-stimulating factor (CSF). Differentiation of macrophage progenitor cells into macrophages (M phi) and expression of various markers were kinetically assessed. The proportion of M phi increases from approximately 4% in freshly isolated BMC to 100% after 7-8 days of cell culture. These cells, termed bone marrow cell-derived macrophages (BMDM phi), adhere to and spread on plastic surface; exhibit M phi morphology; stain intensely for nonspecific esterase; are able to phagocytose latex particles, IgG-sensitized erythrocytes, and C3-coated red cells; and express receptors for IgG and C3. A subpopulation of BMDM phi expresses MHC class II antigens as demonstrated by immunofluorescence using MRC OX6 and MRC OX17 monoclonal antibodies which recognize antigens coded in the I-A or I-E subregion of the MHC, respectively. Collectively, our results show that supernatant from mouse L929 cells supports and is continuously required for proliferation and differentiation of rat BMC into typical M phi, and suggest that mouse CSF cross-reacts with the putative receptor on rat M phi.

The capacity of various types of immunoglobulin for intravenous use to interact with Fc receptors of human monocytes and macrophages.

The capacity of immunoglobulin for intravenous application (IgG-IV) to interact with Fc receptors of human monocytes and macrophages was tested by quantifying the inhibition of phagocytosis of IgG-sensitized erythrocytes. To this end a spectrometric phagocytosis test has been used. When compared with IgG for i.m. use (IgG-IM), all IgG-IV had reduced activity. This reduction was related, in part, to the reduced amount of IgG dimers and polymers in IgG-IV. On a weight basis dimeric IgG and polymeric IgG exerted 6-fold and 14-fold higher activity, respectively, than monomeric IgG. When this difference was corrected for, chemically modified IgG-IV still had significantly reduced inhibitory activity; DEAE-Sephadex-treated IgG and acid-treated IgG had an activity similar to IgG-IM, and PEG-treated IgG showed a slightly reduced activity. Pepsin-treated IgG was greater than 100-fold less active than IgG-IM. The reactivity of IgG-IV with monocyte and macrophage Fc receptors was closely correlated. The most conspicuous differences found were related to the concentration at which IgG was used. Thus, beta-propiolactone-treated IgG and plasmin-treated IgG were found to have significantly reduced activity at concentrations greater than 20 micrograms/ml, but almost normal activity when used at lower concentrations.

Phagocytosis of concanavalin A-treated erythrocytes is mediated by the Fc gamma receptor.

Impaired Fc gamma receptor-mediated phagocytosis has been reported in monocytes from HLA-DR2- and -DR3-positive disease-free individuals compared to normals without these B cell alloantigens. We have noted, however, a decrease in the ingestion of concanavalin A (Con A)-treated rabbit erythrocytes (E-Con A) in the same immunogenetically defined groups (DR2 vs Other: 2.94 +/- 0.84 erythrocytes/monocyte vs 4.16 +/- 1.37, p less than 0.003; DR3 vs Other: 3.35 +/- 1.51 vs 4.16 +/- 1.37, p less than 0.04). These data raised the possibility that carbohydrate-lectin interactions might trigger ingestion mediated by the Fc gamma receptor. To test this hypothesis, we performed receptor modulation and monosaccharide blocking experiments. Modulation of the Fc gamma receptor off the apical cell surface of monocytes by adherence to solid-phase IgG aggregates specifically reduced internalization of E-Con A and IgG-sensitized erythrocytes (EA) to 9.1% and 10.6% of control, respectively (p less than 0.001). Internalization of wheat germ agglutinin-treated erythrocytes, tannic acid-treated erythrocytes, and zymosan was not inhibited. In reciprocal modulation experiments using solid-phase Con A, no effects on phagocytosis of any particle was observed. alpha-Methyl mannoside, 0.1 M in PBS, did not inhibit the internalization of EA but blocked ingestion of E-Con A by 97% (p less than 0.001). Other monosaccharides had little or no effect on the ingestion of any of the phagocytic probes. These data demonstrate that a mechanism integrally involving the Fc gamma receptor mediates the ingestion of E-Con A by human monocytes. This Fc receptor has an oligosaccharide(s) with an exposed mannose which may be functionally significant. Whereas the mannose moiety does not play a crucial role in the interaction of the Fc gamma receptor with the Fc portion of IgG, engagement of the receptor via mannose can initiate internalization. Our findings raise the possibility that nonimmune functions may utilize classical immune system receptors through carbohydrate interactions. Furthermore, the ability of the Fc gamma receptor to trigger internalization is defective in HLA-DR2 and -DR3 normals, whether the receptor is ligated at its classical ligand-binding site or by way of its carbohydrate moieties.

Altered phagocytosis by monocytes from HLA-DR2 and DR3-positive healthy adults is Fc gamma receptor specific.

Fc gamma receptor-dependent mononuclear phagocyte system (MPS) clearance of opsonized erythrocytes is prolonged in healthy adults with the class II alloantigens HLA-DR2, DR3, or DQw1, despite normal receptor-specific Fc ligand binding by monocytes in these groups. To investigate the basis for the MPS dysfunction, we determined the phagocytic capacity of blood monocytes from 66 disease-free adults and analyzed the data according to the HLA type of the subjects. The data demonstrate decreased phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes from HLA-DR2, DR3, or DQw1-positive subjects compared with normals without these B cell alloantigens (2.87 +/- 0.83 erythrocytes/monocyte vs 3.87 +/- 1.05, p less than 0.004; 3.01 +/- 0.94 vs 3.87 +/- 1.05, p less than 0.02; 3.18 +/- 0.89 vs 3.87 +/- 1.05, p less than 0.02, respectively). Because HLA-DR2 and DQw1 are in linkage disequilibrium, we compared EA phagocytosis in subjects with DQw1 alone to normals without HLA-DR2, DR3, or DQw1. Among subjects positive only for DQw1, no significant decrease in phagocytosis could be seen (3.46 +/- 0.95 vs 3.87 +/- 1.05, p = NS). To determine whether these differences represented an Fc receptor-specific dysfunction or a more generalized decrease in phagocytic activity, we compared the quantitative phagocytosis of EA with that of neuraminidase-treated erythrocytes (EN), which is Fc receptor independent and beta-glucan receptor mediated. No segregation of phagocytic capacity for EN by HLA class II phenotypes could be demonstrated (DR2, 2.68 +/- 1.30 erythrocytes/monocyte; DR3, 2.95 +/- 1.30; DQw1, 2.84 +/- 1.15; others, 3.06 +/- 1.14). Our data indicate that the decrease in phagocytosis by blood monocytes from normal individuals with HLA-DR2 or DR3, the class II alloantigens associated with systemic lupus erythematosus and other autoimmune diseases, is specific for the Fc receptor-mediated process. This alteration of Fc receptor function among immunogenetically defined individuals may contribute to their predisposition to autoimmune disease. These differences may also apply to other Fc receptor-initiated cellular functions.

Size and charge heterogeneity of murine IgG-binding factors (IgG-BF).

Size and charge of murine IgG-binding factors (IgG-BF) were determined. Four different sources were used to produce the factors: a) cells of a T cell hybrid (T2D4) constitutively secreting IgG-BF upon incubation in serum-free medium, b) T2D4 cells incubated with mouse monoclonal IgG1 antibody in order to induce in vitro the production of isotype-specific IgG1-BF, c) T2D4 cells induced in vivo by passage as ascites in nude mice and incubated in serum-free medium, and d) in vivo alloantigen-activated T cells (ATC) incubated in serum-free medium. IgG-BF were affinity purified on Sepharose beads coated with rabbit or mouse IgG and identified by their biologic activities, i.e., inhibition of in vitro secondary IgG antibody production to SRBC and inhibition of rosette formation between Fc gamma receptor-positive spleen cells and rabbit IgG-sensitized erythrocytes. IgG-BF produced by either of these cell sources was found to be heterogeneous in both size and charge. In each case, IgG-BF activities were recovered in three fractions of apparent Mr-74,000 to 78,000, 35,000 to 40,000, and 19,000 to 23,000-and in four fractions of pI-4.7 (or 5.3, depending on experimental conditions), 6.5, 7.7, and 8.4. Moreover, IgG-BF translated in vitro from T2D4 poly A RNA by using rabbit reticulocyte lysate exhibited the same heterogeneity. Thus, IgG-BF contain different proteins exerting similar biologic activities.

A rapid and sensitive method allowing photometric determination of erythrophagocytosis by mononuclear phagocytes

A spectrometric assay for assessing erythrophagocytosis by mononuclear phagocytes is described. It is based on the haemoglabin-catalyzed conversion of a benzidine derivative into a coloured product in the presence of H 2O 2 (pseudoperoxidase activity). The assay is set up in microtitre plates, and following an uptake phase and removal of non-ingested erythrocytes, pseudoperoxidase activity is measured in detergent lysates of phagocytes, using an ELISA reader photometer. Various detergents and substrates were evaluated. SDS was found to be the most suitable detergent. Diaminobenzidine (in PBS, pH 7.4) was the substrate of choice for enumerating ingested erythrocytes in a range from 10 4 to 5−8×10 5 sheep erythrocytes. Ortho-tolidine (in acetate buffer, pH 5.5) could be used in a range from 2×10 3 to 2×10 5 sheep erythrocytes. The results obtained with human peripheral blood monocytes or monocyte-derived macrophages and IgG-sensitized sheep erythrocytes correlated well with those obtained using 51Cr-labelled, IgG-sensitized erythrocytes.

Mapping the functional topography of Fcγ with monoclonal antibodies: localization of epitopes interacting with the binding sites of Fc receptor on human K cells

A panel of monoclonal antibodies (mAb) specific for the C gamma 2, C gamma 3 or inter C gamma 2/C gamma 3 domain epitopes was tested for inhibition of antibody-dependent cellular cytotoxicity (ADCC) specific for anti-D IgG-coated erythrocytes. Significant inhibition of ADCC was demonstrable for some antibodies having specificity for C gamma 2 or C gamma 3 domain epitopes, while others gave no inhibition. Fab fragments of a representative C gamma 2-specific antibody (A55) and C gamma 3-specific antibody (x3a8) retained their inhibitory capacity in lymphocyte-mediated ADCC, but only A55 Fab inhibited monocyte-mediated lysis. Furthermore, the Fab portion of A55 completely abolished the complement-dependent enhancement of ADCC mediated by concanavalin A-stimulated cells, while x3a8 Fab had no effect in this system. On the other hand, x3a8 Fab inhibited the binding of anti-D IgG-sensitized erythrocytes to lymphocytes while A55 Fab did not influence this latter interaction. The results suggest that C gamma 2 domain-FcR interaction is essential for the triggering of lytic process both in lymphocyte and in monocyte-mediated ADCC, while C gamma 3 domain has no role in the latter but is responsible for the appropriate contact between effector lymphocytes and target cells. A site in the region of Lys274 appears to be critical for triggering of both lymphocyte and monocyte-mediated ADCC.

Phorbol esters enhance spontaneous cytotoxicity of human lymphocytes, abrogate Fc receptor expression, and inhibit antibody-dependent lymphocyte-mediated cytotoxicity.

Phorbol esters with tumor promoter activity enhance the spontaneous cytotoxicity of human lymphocytes against a variety of target cell lines, with an efficiency that correlates with their potency as tumor promoters or skin irritants. Analysis of surface marker expression of the lymphocytes cytotoxic after treatment with phorbol ester identified the cytotoxic cell subset as that containing natural killer cells. Although gamma-interferon (IFN gamma) is produced by T cells treated with phorbol esters, IFN gamma is probably not the mediator of enhancement of natural killer cell activity, because anti-IFN gamma antibodies failed to block this enhancement. Spontaneous cell-mediated cytotoxicity is inhibited when phorbol esters are present during the cytotoxic assay, but is enhanced when the effector cells are pretreated with these agents. On the other hand, antibody-dependent cytotoxicity mediated by lymphocytes is inhibited by phorbol ester pretreatment of the effector cells or by phorbol esters present during the cytotoxic assay. Treatment of lymphocytes with phorbol esters at 37 degrees C, but not at 4 degrees C, completely abrogates in 1 to 2 hr the expression of the receptor for the Fc fragment of IgG, as detected by rosette formation with IgG-sensitized erythrocytes and by reactivity with anti-Fc receptor antibodies. The inhibition of antibody-dependent cytotoxicity by phorbol esters is probably secondary to their effect on the Fc receptor.

The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies.

We compare five monoclonal antibodies ( B73 .1, 3G8 , Leu- 11a , Leu- 11b , and VEP13 ) that react with natural killer (NK) cells and polymorphonuclear cells (PMN). We show that all of these antibodies are directed against and inhibit the functional properties of the receptor for the Fc portion of IgG (FcR). Modulation of the FcR on NK cells after reaction with immune complexes induces the disappearance of the antigen(s) recognized by each of the five antibodies. Conversely, the antibodies block binding of IgG-sensitized erythrocytes to the NK cells and PMN and inhibit their ability to mediate cytotoxicity against antibody-sensitized tumor target cells. By using two-color immunofluorescence techniques, we characterize directly the lymphocyte population recognized by these antibodies and show that it is a homogeneous subset that does not bear markers of either B or T cells, with the exception of the 33,000 dalton antigen characteristic of suppressor/cytotoxic T cells present in 20 to 50% of the cells, and the 45,000 dalton receptor for sheep erythrocytes present on 80 to 90% of the cells. The phenotype of the cells reacting with the monoclonal antibodies corresponds to that of NK cells. Cross-competition experiments indicate that these antibodies detect at least two distinct epitopes on FcR, one ( B73 .1) preferentially expressed on NK cells and one or more ( 3G8 /Leu- 11a /Leu- 11b / VEP13 ) preferentially expressed on PMN. The lack of reactivity of these antibodies with B cells suggests that human B cells bear a different FcR from that on NK cells and PMN.

Modulation of mononuclear phagocyte function by intravenous gamma-globulin.

To assess the effects on mononuclear phagocyte function of i.v. gamma-globulin treatment in idiopathic thrombocytopenic purpura, we examined in vivo and in vitro mononuclear phagocyte function in 11 patients before and after therapy. All patients, both splenectomized and non-splenectomized, demonstrated a prolongation of in vivo clearance of autologous IgG-sensitized erythrocytes (p less than 0.01). Concurrent in vitro assessment of blood monocyte function showed decreased IgG-sensitized erythrocyte (EA) rosette formation (mean +/- SD: 31.6% +/- 8.2 vs 24.5% +/- 9.5; p less than 0.03) and decreased affinity of Fc receptor-specific IgG oligomer binding (9.9 +/- 16.3 vs 1.8 +/- 2.1 X 10(8) M-1; p less than 0.008), but no consistent change in the estimate of the maximum number of binding sites. Phagocytosis of two different EA probes was decreased (EhuA:0.49 +/- 0.26 vs 0.25 +/- 0.14 erythrocyte/monocyte/hr; p less than 0.02, EoxA: 1.76 +/- 0.66 vs 1.27 +/- 0.67 erythrocyte/monocyte/hr, p less than 0.05). The change in in vivo mononuclear phagocyte system clearance was significantly correlated with the change in the association constant for oligomer binding (r = 0.98, p less than 0.05). These data demonstrate that i.v. gamma-globulin infusions induce alterations of mononuclear phagocyte function that are not dependent on the presence of autologous serum containing infusate. The change in apparent Fc receptor affinity rather than receptor number may reflect an altered Fc receptor population with different binding properties.

Localization of the binding site on IgG for solubilized placental Fc gamma receptor.

Placental Fc gamma R (FcR) inhibited the rosette formation between monocytes and rabbit IgG-sensitized erythrocytes (EA), whereas the rosette formation with granulocytes was not impaired. Staphylococcal protein A (SpA) inhibited the rosette formation with both cell types. Results obtained in absorption and agglutination experiments showed that SpA blocked the binding of FcR to IgG, and Cl did not. Furthermore, FcR did not interfere with the binding of SpA to IgG, whereas C1 affected this binding. FcR apparently bind to the C gamma 3 region. Since FcR inhibited the binding of EA to monocytes, the monocyte FcR binding site is probably also located within the C gamma 3 region.

Activation of macrophages by in vitro allostimulated T cells.

Murine peritoneal macrophages, parabiotically co-cultured with combinations of in vitro H-2 sensitized thymus-derived lymphocytes obtained from drug-pretreated mice, possessed an increased cytotoxicity against alloantibody-coated target cells. This heightened activity appeared to be accentuated by and dependent on T-cell synergy. After 5 days of in vitro allosensitization at 37 degrees C, cortisone-resistant thymocytes allosensitized in combination with cyclophosphamide-pretreated splenic T cells released molecules that produced strong antibody-dependent macrophage-mediated cytotoxicity (ADCC). This enhanced ADCC correlated with increased macrophage rosetting with IgG-sensitized erythrocytes. These heightened activities resulted from soluble mediators released by the activated T cells which diffused across a 0.22-microns Millipore filter and were not dependent on lymphocyte-macrophage contact. Evidence that these molecules originated from the highly enriched T-cell populations and were not synthesized de novo by macrophages was supported by results of pretreatment with protein and RNA synthesis inhibitors. Evidence that soluble Fc receptors released from the alloactivated T cells were responsible for the increased macrophage EA binding and ADCC was obtained in affinity chromatography experiments in which activity could be depleted by passage over a Sepharose-Fc-coupled column and recovered in the column eluate.

Effects of castration and sex hormones on immune clearance and autoimmune disease in [formula omitted] and [formula omitted] mice

The clearance of erythrocytes sensitized with IgG was studied in MRL Mp - lpr lpr (MRL- lpr) and MRL Mp -+/+ (MRL-+/+) mice, which spontaneously develop autoimmune disease. In both strains, an age-dependent decline in clearance of IgG-sensitized erythrocytes was found. Impaired clearance occurred at an earlier age in MRL- lpr mice than in MRL-+/+ mice, correlating with the relative severity of autoimmune disease in these strains. Androgen treatment improved clearance in MRL-+/+ mice but not in MRL- lpr mice, even though autoantibody levels, renal function, and survival were improved. These results suggest that the beneficial effects of androgen on autoimmune disease are not due solely to improved clearance of immune complexes. Castration followed by estrogen administration did not influence immune clearance or autoimmune disease in MRL- lpr mice. These results indicate that impaired immune clearance is a common feature in several autoimmune mouse strains. However, the effects of sex hormones on immune clearance and autoimmune disease may be dissociated in some strains.

On the Mechanisms of Enhanced Monocyte and Neutrophil Cytotoxicity in Severe Psoriasis

Monocyte and neutrophil function assessed as antibody-dependent cell-mediated cytotoxicity (ADCC) using IgG-sensitizing human erythrocytes as target cells was enhanced in patients with severe psoriasis when compared to healthy controls. We found significant correlation between increased monocyte ADCC and increased neutrophil ADCC, No differences in basal cAMP levels and cAMP responses during initiation of the ADCC reaction was observed between psoriatics and normals. Also degranulation determined as lysozyme release during ADCC was normal. In contrast, the increase in ADCC was significantly correlated to an enhanced hexose monophosphate shunt activation in the effector cells during the cytotoxic reaction. Activity of enzymes responsible for the respiratory burst was not altered in psoriasis since superoxide production after stimulation with phorbol myristate acetate was normal. Likewise, oxygen consumption and degranulation following phagocytosis of opsonized zymosan particles in neutrophils was found normal in psoriasis. Since monocytes showed increased binding of IgG-sensitized erythrocytes these data indicate that the enhanced monocyte and neutrophil ADCC is caused by an enhancement of the respiratory burst possibly induced by increased Fc receptor activity.

Heterogeneity of human lymphocyte Fc receptors: Studies using heat-aggregated and antigen-complexed IgG from human, rabbit, guinea pig, horse and goat

We have studied the ability of human peripheral blood lymphocytes (HuPBL) 4 to interact with IgG from several animal species. Three functions or activities that are reported to depend on an interaction between complexed IgG and HuPBL receptors (R) for the Fc piece of IgG (FcγR) were compared: (1) antibody-dependent cell-mediated cytotoxicity (ADCC); (2) binding of heat-aggregated IgG (aggG); and (3) rosette formation with IgG-sensitized erythrocytes [RBC-A(γ)]. IgG (and IgM) antibodies to chicken erythrocytes (CRBC) were purified from the sera of the following species after injection with CRBC stroma: (1) horse (Ho); (2) goat (Go); (3) rabbit (Ra); and (4) guinea pig (Gp). Good IgG-agglutinating antibody titers were obtained from each injected species. Using 51Cr-labeled CRBC targets and HuPBL effector cells, only Ra anti-CRBC IgG gave good ADCC at high dilutions. Ho and Go anti-CRBC (IgG) failed to give AD̄CĊ, and Ḡp anti-CRBC (IgG) gave approx. 30% of the level of kill as Ra. Ra Fab' 2 fragments of IgG antibody failed to produce ADCC. Treatment of HuPBL with Ra anti-lymphocyte serum (ALS) almost totally ablated ADCC, whereas HoALS failed to alter ADCC. Pretreatment of HuPBL with aggG showed that Ra or Hu aggG gave essentially equal inhibition of ADCC, Gp gave approx. 30% of the degree of inhibition as Hu and Ra, and Ho or Go aggG had essentially no effect of ADCC. These results confirmed the following order of ability of IgG to interact with HuPBL ADCC killer (K) cells: (Hu ⪖)Ra > Gp ⪢ Ho, Go. The data suggest that Gp IgG interacts with only a subpopulation (≈ 30%) of HuPBL K cells. The binding of aggG to total HuPBL failed to strictly correlate with the ADCC results or with the results of rosette formation between total HuPBL and CRBC-A(γ). The observations suggest that there is a heterogeneity of FcγR between K and non-K cell subpopulations of HuPBL both in terms of the type of complexed IgG they are able to bind, and in terms of the species of origin of the IgG. The data also support contentions that FcγR that bind RBCA(γ) complexes differ from those that bind aggG.

Histochemical patterns of human T lymphocyte subpopulations with nonspecific esterase staining.

Alpha-naphthyl-acetate-esterase (ANAE) activity was histochemically demonstrated in human T lymphocytes and their subpopulations. Examination of T lymphocytes bearing Fc receptors for IgM showed the presence of circumscribed deposits of esterase activity, while T cells with FC receptors for IgG showed of predominantly diffuse granular pattern. The difference in the topography of the enzymatic activity was shown to be related to cellular activation resulting from exposure to IgG-sensitized bovine erythrocytes or to strong polyclonal activation by lectin mitogens. The ANAE stain also characterized monocytes, which exhibited an intense nongranular cytoplasmic stain. Non-T cells showed no detectable ANAE activity when tested by this method.

Binding of IgG-sensitized erythrocytes by mitral and aortic cardiac valves: A possible clue to the pathogenesis of immune valvular heart disease

The precise pathogenesis of immunologically mediated valvular heart disease is not understood. In this paper we demonstrate the presence of binding activity for the Fc fragment of IgG on sections of human cardiac valves. Binding activity was demonstrated by the specific adherence of IgG-coated sheep erythrocytes (SRBC); SRBC or SRBC coated with IgM antibody were not bound. Moreover, binding activity was greatest on the valves of the left heart and less on valves of the right heart. The rank order of binding activity was mitral, aortic, tricuspid, and pulmonic. Binding activity was selectively inhibited by IgG in solution but not by the (Fab′) 2 fragment of IgG. The IgG-Fc binding activity of cardiac valves is distributed in the subendothelial intercellular ground substance of the base, stalk, and tip of the valve. Cardiac valvular binding sites were sensitive to preincubation of the sections with hyaluronidase but resistant to collagenase suggesting the binding sites reside in noncollagenous ground material. The presence of IgG-Fc binding sites on cardiac valves may help explain the pathogenesis of immune valvular heart disease.

Clearance of sensitized erythrocytes in NZB/NZW mice. Effects of castration and sex hormone treatment.

The clearance of particulate immune complexes consisting of erythrocytes sensitized with IgG or complement was investigated in (NZB x NZW)F1 (B/W) mice. Treatment of castrated B/W mice with androgen or estrogen was able to modulate this clearance. Young (3-month-old) male and female B/W mice cleared IgG-sensitized mouse erythrocytes rapidly, whereas older males (13 months) and females (7 months) showed a marked impairment in their ability to clear these cells. In addition, erythrocytes sensitized with complement in the absence of antibody were cleared within 5 min in young B/W mice. Older mice showed a greater and more rapid clearance rate of these cells. Castrated female B/W mice treated with androgen implants from three weeks of age showed improved clearance of IgG-sensitized erythrocytes at 7 months, whereas estrogen-treated male mice showed delayed clearance. These results suggest an age-dependent defect in the clearance of IgG-sensitized particles, perhaps due to diminished levels of serum complement and/or saturation of Fc receptors. In addition, there is an alteration in the clearance of complement-sensitized erythrocytes which may be related to changes in macrophage activity or enzyme inactivators of C3 and C4. The possible mechanisms responsible for the hormonal modulation of clearance are discussed in relation to the known ability of these hormones to influence autoimmune diseases.

Mechanisms of decreased antibody-dependent cytotoxicity mediated by monocytes and neutrophils in atopic dermatitis

By using IgG-sensitized erythrocytes as target cells, we found the antibody-dependent cytotoxicity mediated by monocytes and neutrophils to be depressed in atopic dermatitis. This change was accompanied by a reduction of phagocytosis of target cells by the effector cells, and also by a decreased lysozyme liberation from, and hexose monophosphate shunt activation in, the effector cells during the cytotoxic reaction. However, the binding of target cells to effector cells remained normal. In individual patients, cytotoxicity and phagocytosis were equally depressed. No relation was found between cytotoxicity and lysozyme liberation in atopic dermatitis, but a relation between cytotoxicity and hexose monophosphate shunt activity was highly significant. These results suggest that the decreased cytotoxicity mediated by monocytes and neutrophils in atopic dermatitis is caused by a diminished production of oxygen radicals during the respiratory burst. This reduced respiratory burst was not due to any defect in the enzymes responsible for this process, since the superoxide production of monocytes and neutrophils stimulated with phorbol myristate acetate was found normal in atopic dermatitis. It therefore seems that although binding of target cells to effector cells was normal, the signals created by the binding process were defective in activating monocytes and neutrophils in atopic dermatitis.