IgA-induced eosinophil degranulation

Eosinophils play an important role as effector cells in allergic, parasitic, and other conditions. The mechanism(s) by which eosinophils mediate their effector functions was studied by incubation of human normodense eosinophils with Sepharose beads coupled to various Ig isotypes as targets. Controls included eosinophils incubated alone or incubated with uncoated beads, human serum albumin-, or OVA-coated beads. An eosinophil granule protein, the eosinophil-derived neurotoxin (EDN), was measured as an indicator of eosinophil degranulation. Eosinophils released eosinophil-derived neurotoxin when incubated with Sepharose beads coupled to Ig of the IgG or IgA isotypes, as well as IgA-Fc fragments. Mixtures of IgG and IgA on beads did not act synergistically. Secretory IgA (sIgA) provided the most potent signal for eosinophil degranulation and was two to three times more potent than IgG. Furthermore, 2 to 17% of the normodense eosinophils bound to IgG- or IgA-coated beads, whereas 24 to 27% of the eosinophils bound to sIgA-coated beads. Thus, sIgA may be the principal Ig mediating eosinophil effector function at mucosal surfaces in helminth infections and hypersensitivity diseases, especially bronchial asthma.     

F(ab)'2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB2) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)2-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)2. This binding could interfere with the role of C3a and C5a in inflammation.     

Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab′)2 fragments

This study evaluated an automated immunoassay for bovine lactoferrin (LF) in dairy products based on latex beads coated with F(ab')2 fragments. Methods: F(ab')2 fragments were obtained by pepsin digestion of rabbit anti-bovine LF (IgG fraction) and polystyrene latex beads were coated with the F(ab')2 fragments. We used the beads to develop a rapid and homogeneous light scatter immunoassay employing an autoanalyzer (the Automated Latex assay). The Automated Latex assay was easy to perform and could rapidly determine bovine lactoferrin in lactoferrin-supplemented products. It was sensitive enough for testing products and showed good precision.     

Eosinophil-particle interactions: a model system for study of cellular adherence and activation

In its role as an effector capable of killing large multicellular parasites, the eosinophil must be especially adapted for dealing with noningestible surfaces. A model system of Sepharose beads coated with serum protein or concanavalin A (Con A) has been used to study interactions between guinea pig peritoneal exudate eosinophils and noningestible particles. A small percentage of eosinophils were adherent to serum treated Sepharose; however, many cells were adherent to Con A-Sepharose. Adherence to Con A-Sepharose was decreased by pretreatment with 1 mM alpha-methylmannoside (alpha-MM). As compared to resting eosinophils, incubation of eosinophils with serum-treated Sepharose led to activation of oxidative metabolism as indicated by an eight-fold increase in superoxide anion production and an approximately threefold increase in quantitative leukocyte iodination. Eosinophils which were adherent to Con A beads could not be activated by either phorbol myristate acetate (PMA) or preopsonized zymosan. However, if adherence was reduced by preincubation with alpha-MM, PMA was able to activate the eosinophils. Neither soluble Con A nor Sepharose beads interfered with the assay of superoxide anion. These studies demonstrate the utility of Sepharose beads for studying interactions between eosinophils and noningestible particles.     

Ultrastructural localization of immunoglobulin G and complement C9 in the brain stem and spinal cord following peripheral nerve injury: an immunoelectron microscopic study

Summary The ultrastructural localization of immunoreactivity for immunoglobulin G (IgG), F(ab′)2 and complement C9 was examined with preembedding immunoelectron microscopy in the hypoglossal nucleus and gracile nucleus as well as in the L4 spinal cord dorsal horn 1 week following hypoglossal or sciatic nerve transection, respectively. Only a few scattered immunoreactive profiles were observed on the unoperated side. On the operated side, IgG and F(ab′)2 immunoreactivity was present in the membranes of all reactive microglial cells observed. In addition, the cell membrane of some hypoglossal motoneurons showed IgG immunoreactivity. Complement C9 immunoreactivity was present in the cytoplasm of all reactive microglial cells examined. In addition, there was diffuse C9 immunoreactivity in motoneuron perikarya ipsilateral to nerve injury as well as in cell membranes in the neuropil, some of which could be identified as neuronal. Our interpretation of these findings is (1) that peripheral nerve injury results in binding of IgG to reactive microglia, as well as to some axotomized neurons, and (2) that C9 is synthesized by reactive microglia in response to axon injury and is also associated with axotomized motoneurons. These findings suggest that IgG and complement C9 are involved in microglia-neuron interactions after peripheral nerve injury.     

Neutrophil C3bi receptors: formation of membrane clusters during cell triggering requires intracellular granules

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.     

Bactericidal but not nonbactericidal C5b-9 is associated with distinctive outer membrane proteins in Neisseria gonorrhoeae

In this study, we examined the bacterial constituents associated with the complement C5b-9 complex in detergent extracts from serum-treated Neisseria gonorrhoeae (GC). 125I surface-labeled GC were incubated in 10% serum, were washed, and were solubilized in the zwitterionic sulfobetaine detergent SB12. Immunoprecipitation of 125I-GC from the extract with anti-C5 Sepharose was followed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of immunoprecipitated material. Polyacrylamide gel analysis of surface-labeled 125I-GC showed prominent bands for proteins I and III for both serum-resistant GC strain 6305 and serum-sensitive GC strain 7189. These same bands were visible with similar intensity in the SB12 extracts from presensitized and non-presensitized 6305 and 7189 after serum incubation. For those organisms bearing bactericidal C5b-9 (6305 + IgG and 7189 +/- IgG), additional distinctive bands immunoprecipitated with antibody to C5 Sepharose. These components were of 93,000, 44,000 40,000, and 15,000 daltons for 6305 + IgG, and were of 90,000, 50,000, 44,000, and 19,000 daltons for 7189 +/- IgG. Nonbactericidal C5b-9 extracted from the surface of 6305 incubated in serum, but not sensitized with antibody, was not associated with these distinctive proteins. However, this nonbactericidal C5b-9 did have a different pattern of associated bacterial surface constituents from that observed in control samples incubated with antibody to human serum albumin, which were similar to those with nonserum-incubated organisms. These studies support our earlier experiments which demonstrated that C5b-9 is in a different molecular configuration on the surface of serum-resistant GC from that on the surface of serum-sensitive GC or resistant GC rendered sensitive with bactericidal antibody.     

Regulatory effect of cytokines on eosinophil degranulation

We tested the effects of different cytokines on IgA- and IgG-induced eosinophil degranulation in vitro to determine the potential interaction between eosinophils and mononuclear cells. Purified normodense eosinophils were incubated with cytokines (including rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, IFN-gamma, granulocyte-macrophage CSF stimulating factor (GM-CSF), and TNF) for 1 to 3 h after which Ig-coupled Sepharose 4B beads were added as targets and the mixtures were incubated with the eosinophils at 37 degrees C for 4 h. The Ig used were secretory IgA (sIgA), serum IgA and IgG, and myeloma IgA and IgG. The release of eosinophil-derived neurotoxin (EDN) was measured by RIA as an index of degranulation. rIL-5 was the most potent enhancer of Ig-induced degranulation and increased EDN release by 48% for sIgA and 136% for IgG. The effect of rIL-5 appeared as quickly as 15 min after incubation of eosinophils, sIgA beads and IL-5. GM-CSF and rIL-3 also enhanced Ig-induced EDN release but less potently than rIL-5. GM-CSF and rIL-5 by themselves induced a small but significant release of EDN from eosinophils in the absence of Ig-coated beads; rIL-3 did not. However, IFN-gamma suppressed sIgA-induced EDN release by 23%. The other cytokines did not have any effect on eosinophil degranulation. These results suggest that cytokines which induce eosinophil differentiation and proliferation during hematopoiesis also enhance the effector function of mature eosinophils and that IFN-gamma partially down-regulates eosinophil degranulation.     

Osmotic stress and the freeze-thaw cycle cause shedding of Fc and C3b receptors by human polymorphonuclear leukocytes

A major problem in the cryopreservation of human polymorphonuclear leukocytes (PMN) is the loss of phagocytic function in cryopreserved cells. This is not a problem with cryopreserved monocytes. To study the reasons for this difference in detail, PMN and monocytes were either osmotically stressed in hypertonic media or were frozen to various temperatures. Cells were then returned to conditions of physiologic osmolarity and temperature. All cells remained viable. However, the ability of PMN to phagocytize bacteria and to bind sheep erythrocytes (E) opsonized with IgG, C3b, or C3bi decreased sharply after exposure to media of 600 mOsM or greater and after freezing to -1.5 degrees C. In contrast, monocytes were unaffected until a concentration of 1500 mOsM or a freezing temperature of -5 degrees C was exceeded. To determine whether the functional losses of surface receptor activity in PMN resulted from a loss of receptors from the membranes or from inactivation or internalization of receptors, opsonized E were incubated in the supernatants from stressed PMN. On subsequent incubation with healthy PMN, these E made fewer rosettes than control opsonized E. The inhibitory effect of the supernatants on rosetting of IgG-sensitized E could be removed by preincubation with IgG bound to Sepharose 4B. Immunoprecipitation of C3b and C3bi receptors from surface-iodinated, osmotically stressed, and control PMN suggested that about 50% of cell surface complement receptors were lost from the cell surface during osmotic stress. These experiments suggest that receptors for IgG and C3 are extruded from PMN cell membranes as a result of hyperosmotic stress, which is associated with the freeze-thaw cycle. This may be an early event in the functional damage done to PMN during attempts at cryopreservation.     

Leukocyte complement: a possible role for C5 in lymphocyte stimulation

The results presented here show that Fab' antibody fragments directed to complement proteins C5, C6, and C7 inhibit lymphocyte stimulation in mixed lymphocyte culture (MLC) by up to 65%, as determined by decreased incorporation of 3H-thymidine. Lymphocyte stimulation induced by PHA-mitogen was also inhibited up to 100% by anti-C5 Fab'. Specificity of these reactions was established by the findings that goat anti-C5 or murine hybridoma anti-C5 both inhibited MLC; the inhibitory activity of anti-C5 Fab' was absorbed with highly purified C5 (but not with C3), and antibody directed to C3 did not inhibit lymphocyte stimulation by MLC or PHA. The effects of anti-C5 were exerted in a nontoxic manner. Cleavage of lymphocyte associated C5 with factor B (Bb) or with trypsin resulted in stimulation of lymphocyte thymidine incorporation. Purified C5a was found to induce lymphocyte stimulation in serum-free medium in pulse-chase types of experiments. Anti-C6 and C7 Fab' also inhibited lymphocyte stimulation induced in one-way MLC. These results suggest that C5, C5a, and/or C6 and C7 may play a role in triggering of lymphocyte blastogenesis.     

Inhibition of DNA replication and DNA polymerase alpha activity by monoclonal anti-(DNA polymerase alpha) immunoglobulin G and F(ab) fragments

The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro: I. Effects of anti-C4 on cultured guinea pig peritoneal cells

Suppression of the fourth component of complement in vitro was examined by exposing cultured guinea pig peritoneal cells to anti-C4 alloantisera or to control sera. We found that in contrast to similar experiments in vivo, C4 production could be suppressed by specific anti-C4 antisera over a wide range of doses with complete regularity. The suppression was not permanent, however, and postsuppression stimulation of C4 was frequently seen after recovery from suppression. Production of C2 and β-glucuronidase were also measured in these experiments. C2 was not suppressed by the anti-C4 treatment but stimulation of C2 was seen in those instances where C4 production was stimulated. β-Glucuronidase was neither suppressed nor stimulated. Purified IgG₁ IgG₂ and (Fab′)₂ fragments were as effective in causing suppression and postsuppression stimulation as whole antibody preparations. This suggests that complement activation, Fc receptors, and complement receptors do not play a significant role in these in vitro phenomena.     

Fc epsilon RI-mediated association of 6-micron beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling

Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6- micron beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-micron beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand- coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-micron beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-micron bead/cell interface, including p53/56lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells.     

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.     

T lymphocyte aggregation with immobilized anti-TCR-antibodies is dependent upon energy and microfilament assembly

An assay has been developed to quantitate the binding of beads coated with anti-T cell receptor (TCR) monoclonal antibodies (MoAb) to T lymphocytes. The Ab used were a hamster MoAb, 145.2C11 (2C11), directed against the epsilon chain of the CD3 complex of the murine TCR, and a murine MoAb, F23.1, directed against the V beta 8-encoded determinant of the alpha/beta heterodimer of the TCR. Ab were adsorbed onto polystyrene beads and the beads labeled with [125I]bovine serum albumin [( 125I]BSA). The labeled, Ab-coated beads were mixed at 4 degrees C with murine, cloned T-helper (Th) cells and contact between beads and cells was promoted by centrifugation. The mixtures were incubated at 37 degrees C for 10-20 min, and unbound beads were separated from cell-bound beads by Percoll gradient centrifugation. Beads coated with anti-TCR Ab formed stable conjugates with Th cells; an average of 6-10 2C11 Ab-coated beads/cell, or 10-15 F23.1 Ab-coated beads/cell was measured under optimal conditions. Beads coated with control Ab (hamster or mouse IgG) did not appreciably bind to the cells. Conjugation with 2C11 Ab-coated beads could be prevented by coating the cells with soluble 2C11 Ab, but not with soluble F23.1 Ab. Blocking the CD3 epsilon chain with soluble 2C11 Ab also reduced conjugate formation with F23.1 Ab-coated beads, suggesting a steric hindrance phenomenon. The extent of conjugation depended on the density of immobilized Ab. Maximum conjugation was observed when 100 micrograms of 2C11 Ab was used to coat 10(6) beads; higher Ab amounts did not further increase binding. Increasing the bead to cell ratio in the mixture increased binding, reaching optimal binding at 300:1, irrespectively of the amount of Ab adsorbed onto the beads. Stable binding of anti-TCR Ab-coated beads to T cells was temperature and energy dependent. It was prevented when glucose was removed from the medium and the glycolysis inhibitor, 2-deoxy-D-glucose was added, or when cells were treated with sodium azide. Conjugate formation was prevented by pretreatment of the cells with cytochalasins, indicating that microfilament assembly was essential. Microtubules were not involved, as vinca alkaloids were without effect. This novel assay system provides a simple means of studying aspects of TCR function including its physical and metabolic regulation.     

Neutrophil adhesion to xenogeneic endothelium via iC3b

Neutrophils are thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. We asked whether the interactions of human polymorphonuclear leucocytes (PMN) with xenogeneic endothelium might be promoted by the binding of natural anti-endothelial antibodies and complement by using porcine aortic endothelial cells (PAEC), human serum, and human PMN in an in vitro model of hyperacute rejection. Pretreatment of PAEC with 10% human serum followed by washing markedly increased PMN adhesion from 15.7 +/- 1.8% to 62.5 +/- 3.6% (p less than 0.001). Complement and anti-endothelial antibodies were necessary for the increase, because heat-inactivated serum or serum depleted of IgM did not significantly increase PMN adhesion to treated endothelium. The induction of increased PMN adhesion to PAEC by human serum was observed within 1 min. The essential role of complement was defined using complement-depleted serum. Ten percent C2-deficient serum did not increase PMN adhesion whereas 10% C5-depleted or 10% C8-depleted serum caused the same increase in PMN adhesion as observed with normal human serum. These results suggested that C3 might play a critical role in enhanced neutrophil adhesion. In fact, PAEC treated with 10% human serum for 15 min and incubated with an F(ab')2 antihuman C3 for 10 min completely abolished the enhanced adhesion. PAEC treated with 10% human serum or C5-depleted serum displayed fluorescence of iC3b whereas monolayers treated with heat-inactivated serum or C2-deficient serum were non-reactive. The enhanced PMN adhesion to serum-treated PAEC was mediated through neutrophil receptors binding iC3b because mAb directed against CD11b/CD18 inhibited the serum-enhanced adhesion of PMN. We conclude that PMN adhesion to endothelium can be significantly enhanced by the endothelial deposition of iC3b.     

The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F(ab) and F(c) fragments

The unfolding and further denaturation of IgG and its F(ab) and F(c) fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F(ab) and F(c) units was retained after fragmentation of the IgG. The F(ab) fragment denatured at approximately 61 degrees C and the F(c) fragment at 71 degrees C. The structural transitions observed in the whole IgG is the sum effect of those determined for the isolated F(ab) and F(c) fragments.     

Immune responses during human schistosomiasis mansoni. XV. Anti-idiotypic T cells can recognize and respond to anti-SEA idiotypes directly

We previously have shown that former patients and patients with active cases of schistosomiasis mansoni have T lymphocytes in their PBMC that proliferate when exposed to immunoaffinity-purified antibodies against Schistosoma mansoni soluble egg antigens (SEA). These T cell anti-idiotypic responses required the participation of adherent cells, but the role of these cells in the response to the Id has been unclear. We now show that chloroquine does not interfere with Id-elicited stimulation of cells from former patients but completely inhibits their response to the SEA. F(ab')2 fragments of anti-SEA Id are stimulatory, and excess normal human IgG does not alter anti-Id responses. Soluble Id F(ab) fragments are not stimulatory, but rather inhibit stimulation by the intact Id from which they were made. Either intact Id or their F(ab')2 fragments can stimulate non-adherent T cells in the absence of adherent cells if an exogenous source of purified or recombinant human IL-1 is supplied. Nonstimulatory F(ab) fragments can stimulate nonadherent cells if they are bound first to Sepharose 4B and presented in conjunction with IL-1. Thus, T cells from former schistosomiasis patients can react with polyclonal anti-SEA-related Id directly. Under these conditions T cell proliferation requires receptor cross-linking and a source of IL-1 but does not require either "processing" of Id or MHC co-presentation.     

Specific antibody promotes opsonization and PMN-mediated killing of phagocytosis-resistant Enterococcus faecium

Many clinical isolates of Enterococcus faecium are resistant to neutrophil (PMN)-mediated phagocytosis and killing in the presence of normal human serum. We have now examined the ability of specific polyclonal rabbit antibodies to promote opsonization and killing of phagocytosis-resistant E. faecium. Immune rabbit serum generated against formalin-killed E. faecium TX0016, a phagocytosis-resistant strain, markedly promoted binding of TX0016 organisms to PMNs and PMN-mediated killing. These effects were dramatically reduced by (a) adsorption of immune serum with E. faecium TX0016, but not by adsorption with a strain of E. faecium susceptible to phagocytosis, and (b) incubation of immune serum with carbohydrate purified from TX0016, but not by incubation with a surface protein extract from TX0016. IgG purified from immune serum was unable by itself to promote bacterial binding to PMNs. However, specific IgG was able to promote binding to PMNs and PMN-mediated killing in the presence of normal human serum as a complement source, as were F(ab')(2) and Fab fragments produced from it, and the alternative pathway of complement was sufficient to promote IgG- and F(ab')(2)-mediated opsonization. PMN complement receptor type 3, but not complement receptor type 1, was involved in bacterial binding to PMNs induced by the combination of F(ab')(2) fragments and normal human serum. These results suggest that opsonization by antibodies potentially directed against bacterial carbohydrate, in conjunction with complement activation, has an important role in the host defense against phagocytosis-resistant E. faecium.     

Induction of IL-1 secretion from human monocytes by Fc region subfragments of human IgG1

Peripheral blood-derived human monocytes and the murine P388D1-monocytes-like cell line are induced to secrete IL-1 when stimulated with Fc region but not F(ab) region subfragments obtained from the cleavage of human IgG1 with papain or pepsin. The portion of the Fc region of IgG1 responsible for stimulation of IL-1 secretion appears to be located within the C gamma 3 domain of the molecule. This hypothesis is supported by the observation that the biologically active pepsin-derived pFc' subfragment is located within the C gamma 3 domain and the long-term papain digests containing predominately Fc' are also active. In contrast, short term papain digests containing mostly intact Fc fragments were found to be unable to induce IL-1 secretion.