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Abstract
Cross-linking receptors for the Fc region of immunoglobulin G (IgG) (FcgammaR) induces tumor necrosis factor-alpha (TNF-a) release; however, there is controversy about release of interleukin (IL)-1beta. The purpose of this study was to investigate the role of endotoxin priming on the ability of monocytes to release these cytokines after FcgammaR cross-linking. Monocytes were incubated with plated or soluble human IgG or albumin with or without endotoxin priming. Monocytes isolated by Percoll, containing low concentrations of endotoxin, and incubated on plated IgG released 4.5 +/- 1.6 ng/ml TNF-alpha and 1.6 +/- 0.6 ng/ml IL-1beta. Monocytes isolated by a ''clumping'' technique released 1.0 +/- 0.4 ng/ml TNF-alpha but no IL-1beta. Priming with endotoxin, which did not affect FcgammaR expression, resulted in augmented release of TNF-alpha (4.3 +/- 1.3 vs. 0.1 +/- 0.0 ng/ml, P < 0.05) and IL-1beta (4.0 +/- 1.0 vs. 0.6 +/- 0.3 ng/ml, P < 0.01) when clumped monocytes were incubated on plated IgG vs. plated albumin.
... FcγR-mediated cytokine production requires the activation of upstream MAP Kinases (Erk, p38 and Jnk) and the transcription factors NFAT, NFκB and AP-1 88,224,277 . ...
... It is already known that signaling through the Fc receptors like CD32 and CD64 can activate macrophages for higher production of TNF-a (41). To rule out this possibility, we next treated anti-B7-1 and anti-B7-2 antibodies with pepsin to remove the Fc portion and used the purified F(ab#) 2 fragments to cross-link B7-1 and B7-2 receptors. ...
Blocking T cell co-stimulatory signals by anti-B7-1/B7-2 mAb is an attractive approach to treat autoimmune diseases. However,
anti-B7-1/B7-2 mAb treatment is known to exacerbate autoimmune diseases through mechanisms not fully understood. Tumor necrosis
factor alpha (TNF-α) and reactive oxygen species (ROS) also play important roles in determining the clinical outcome of autoimmune
diseases. In this study, we demonstrate that the anti-B7-1 and the anti-B7-2 mAbs activate macrophages for higher induction
of TNF-α and other effector responses such as bacterial cytotoxicity and production of ROS. Nuclear factor-kappaB (NF-κB)
was found to be increased with anti-B7-1/B7-2 mAb treatment. Inhibition of NF-κB activity by over-expression of phosphorylation-defective
I-kappaB alpha in anti-B7-1/B7-2 mAb-treated macrophages decreased TNF-α production. These data indicate that anti-B7-1 and
anti-B7-2 mAbs can trigger innate-effector responses in macrophages by activating NF-κB-signaling pathway. Our results suggest
that the B7 molecules are not only essential for induction of adaptive immune responses but also play roles in activation
of innate immune responses.
The review article deals with modern aspects of liver immune status in hepatic insufficiency caused by hepatobiliary disorders. The mechanisms of cytokine effects are substantiated, as well as their action upon clinical course of hepatic insufficiency, and their influence upon development of potential post-surgical complications is discussed.
Objective:
To investigate the role of Fcγ receptors (FcγRs) in osteoclastogenesis and osteoclast function.
Methods:
Bone destruction was analysed in arthritic knee joints of several FcγR-knockout mouse strains. Unfractionated bone marrow cells were differentiated in vitro towards osteoclasts in the absence or presence of immune complexes (ICs) and stimulated thereafter for 24 h with tumour necrosis factor α (TNFα) or lipopolysaccharide (LPS). In addition, mature osteoclasts were stimulated with ICs. Experiments were analysed for osteoclast formation, bone resorption and the expression of FcγRs and osteoclast markers.
Results:
Bone destruction was significantly increased in arthritic knee joints of FcγRIIB-deficient mice. All FcγR classes were highly expressed on osteoclast precursors. Expression of the inhibitory FcγRIIB was similar on mature osteoclasts compared to macrophages, whereas activating FcγR levels were significantly lower. IC stimulation of mature osteoclasts did not affect their number or their bone resorptive capacity. ICs significantly inhibited differentiation of unfractionated bone marrow cells towards osteoclasts, bone resorption and expression of osteoclast markers. In the presence of ICs, osteoclastogenesis of FcγRIIB(-/-) precursors and bone resorption remained inhibited. In contrast, ICs could not inhibit osteoclast formation or bone resorption of FcRγ-chain(-/-) precursors. When IC-inhibited osteoclastogenesis was followed by stimulation with TNFα or LPS, the inhibitory effects of ICs were overruled.
Conclusion:
Activating FcγRs mediate IC-induced inhibition of osteoclastogenesis, which might be overruled in the presence of proinflammatory mediators. This suggests that the balance of FcγR-mediated inflammation, through proinflammatory cytokine production, as well as the direct inhibitory effect of ICs on osteoclastogenesis determines the net effect on bone loss.
Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcgamma receptor (FcgammaR) function, as they are critical mediators of antibody therapy.
The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcgammaR. Murine bone marrow-derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcgammaR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model.
Overnight incubation with R-848 increased FcgammaR-mediated cytokine production and antibody-dependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcgammaRI, FcgammaRIIa, and the common gamma-subunit was increased. Surprisingly, expression of the inhibitory FcgammaRIIb was almost completely abolished. In bone marrow-derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the gamma-subunit in TLR7(-/-) nor MyD88(-/-) cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody.
These results show an as-yet-undiscovered regulatory and functional link between the TLR7/8 and FcgammaR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy.
To review the use of liposomes as a delivery agent in inflammatory arthritis.
The literature on liposomes and liposomal drug delivery for the treatment of inflammatory arthritis was reviewed. A PubMed search of articles in the English-language journals from 1965 to 2007 was performed. The index words used were as follows: "rheumatoid arthritis," "liposomes," and "targeted delivery." Papers identified were reviewed, abstracted, and summarized.
Liposomes have the capacity to be used as delivery and targeting agents for the administration of antirheumatic drugs at lower doses with reduced toxicity. In other areas of medicine, the pace of progress has been rapid. In the case of infectious diseases and cancer, liposomal drug delivery has progressed and developed into commercially viable therapeutic options for the treatment of fungal infections (amphotericin B), or metastatic breast cancer and Kaposi sarcoma (doxorubicin, daunorubicin), respectively. In arthritis, the efficacy of prednisolone-loaded long-circulating liposomes is currently being evaluated in a phase II clinical trial. Liposome's application to arthritis is still in its infancy but appears promising as new patents are filed. With improvements in liposomal formulation and targeted synovial delivery, liposomes offer increased therapeutic activity and improvement in the risk-benefit ratio.
Recent research into synovial targets and improved liposomal formulations continues to improve our capacity to use liposomes for targeted delivery. With time, this approach has the potential to improve drug delivery and reduce systemic complications.
The cross-linking of monocyte Fc gamma R is a potent stimulus for IL-1ra production but does not induce IL-1 beta. However, during systemic infection, IgG-coated bacteria can activate mononuclear phagocytes via both cell wall components and opsonized IgG. Therefore, we analyzed the effect of combinations of Fc gamma R cross-linking and bacterial cell wall components on mononuclear phagocyte IL-1 beta and IL-1ra production. Human mononuclear cells and monocytes were cultured either alone or with combinations of immobilized IgG, LPS, or heat-killed Staphylococcus aureus (HKSA). Cells cultured on immobilized IgG released large amounts of IL-1ra but no detectable IL-1 beta. In response to LPS, mononuclear cells released IL-1ra at 1000-fold lower doses of LPS than was required to induce IL-1 beta. However, when measured in the presence of immobilized IgG, the LPS sensitivity for IL-1 beta release increased 100-fold, whereas IL-1ra release correlated inversely with the LPS dose. Furthermore, HKSA, a nonendotoxin stimulus, affected mononuclear cell IL-1 beta and IL-1ra release similarly. In addition, polymyxin B, a specific endotoxin inhibitor, blocked the LPS, but not the HKSA-induced changes in IL-1 beta and IL-1ra secretion, irrespective of immobilized IgG co-stimulation. In summary, these results suggest that mononuclear phagocyte stimulation with immobilized IgG favors IL-1ra over IL-1 beta production. Conversely, the addition of LPS or HKSA to the Fc gamma R-stimulated cells augments IL-1 beta but suppresses IL-1ra production.
Autoantibodies in the form of immune complexes are known to be crucial mediators in initiating inflammation in a variety of autoimmune diseases. This has been well documented in the anti-collagen II antibody-induced arthritis animal model for a long time now. Recently, in the K/B x N mouse model (the F1 of the TCR-transgenic KRN and the diabetic NOD mice), anti-glucose-6-phosphate isomerase (GPI) autoantibodies have been shown to induce arthritis. Experimental work in the K/B x N model demonstrated key roles of autoantigenic immune complexes activating the alternative pathway of complement, the subsequent association with C5aR and Fc gammaRIII-mediated cell activation and production of the inflammatory cytokines IL-1 and TNF-alpha, finally leading to joint destruction. The presence of high amounts of inflammatory cytokines and matrix-degrading proteases at sites of inflammation obviously put the cytokine-producing macrophages as the next target for investigation in this model. Here, we show that mice depleted of macrophages by clodronate liposome treatment are completely resistant to K/B x N serum-induced arthritis. Reconstituting clodronate liposome-treated mice with macrophages from naive animals could reverse this resistance. Also, we found that deficiencies in the Wiskott-Aldrich syndrome protein and CD40, which are both implicated in macrophage activation, chemotaxis and phagocytosis, are not essential in serum-induced arthritis. Mast cell degranulation was seen in arthritogenic serum-treated mice even in the absence of macrophages, possibly suggesting that mast cell degranulation/activation acts hierarchically before macrophages in the inflammatory cascade of anti-GPI antibody-induced arthritis.
We report evidence that FcR(CD16) on human NK cells are signal-transducing molecules that, upon ligand binding, induce transcription of genes encoding surface activation molecules [IL-2-R(CD25)] and cytokines (IFN-gamma and TNF) relevant to NK cell biology and functions. Homogeneous NK and T cell populations purified from short-term bulk cultures of PBMC with irradiated B lymphoblastoid cell lines were cultured in the presence of FcR ligands (particulate immune complexes or immobilized anti-CD16 antibodies) alone or with rIL-2. Upon 18 h of stimulation, NK cells express Tac, TfR, and 4F2 antigens and produce IFN-gamma and TNF; both effects are synergistically enhanced in the presence of rIL-2, which is itself ineffective. Treatment of NK cells with FcR(CD16) ligands induces accumulation of mRNA for IFN-gamma and TNF and, to a lesser extent, IL-2-R with fast kinetics also in the absence of de novo protein synthesis. rIL-2 and FcR(CD16) ligands synergize to induce mRNA accumulation. mRNA accumulation and transcription of TNF and IFN-gamma genes induced by FcR(CD16) ligands are greater than those induced by rIL-2, and the reverse is true for IL-2-R. The two stimuli do not synergize at the transcriptional level. These observations indicate that the mechanisms through which FcR(CD16) ligands and rIL-2 induce NK cell activation are, in part, distinct. Both operate at the transcriptional level, although other mechanisms are probably induced by the FcR ligand stimulus per se or in combination with other lymphokines and synergize at a post-transcriptional or translational level to enhance NK cell activation.
The Fc receptors for IgG from a human monocyte line (U937) and from highly purified human peripheral blood monocytes were solubilized, purified, and partially characterized. Both sources of cells gave indistinguishable results. Two molecules (or sets of molecules), one of about 72,000 mol wt and the other of 40,000-43,000 mol wt were discerned on autoradiograms of sodium dodecyl sulfate (SDS)-polyacrylamide gels analyzing acid eluates from Sepharose-IgG columns over which detergent lysates of radioiodinated cells had been passed. The larger of the two molecules, p72, accounted for greater than or equal to 90% of the radioactivity. This component was noted to be heterodisperse both by size on SDS gels and by charge on isoelectric focusing gels. The charge heterogeneity, being virtually eliminated by neuraminidase and tunicamycin, was probably due to variable glycosylation. Several lines of evidence indicated that p72 is probably all or part of the Fc receptor: (a) radiolabeling of this molecule using chloroglycouril was blocked by IgG of the Fc receptor; (b) in soluble form this molecule expressed ligand specificity identical to the in situ receptor; (c) the molecule was not recovered from affinity adsorbants bearing proteins that do not bind to the Fc receptors, nor (d) from a human T cell line that does not bear Fc receptors. The smaller of the two molecules isolated, p40-43, is at least in part actin. Its relationship to p72 is not understood.
We have evaluated the capacity of the three major classes of human Fc gamma R to mediate phagocytosis by measuring the ability of adherent phagocytes to internalize erythrocytes coated with anti-Fc gamma R mAb. Five different cell types were studied, freshly purified monocytes, cultured monocytes, alveolar macrophages, freshly purified polymorphonuclear neutrophilic leukocytes, and PMNs cultured in IFN-gamma. Fc gamma RI and Fc gamma RII on whichever cells they were expressed were capable of phagocytosing anti-Fc gamma R mAb-coated erythrocytes. Furthermore, Fc gamma RIII on mononuclear phagocytes, which appears to be a conventional integral membrane protein that spans the lipid bilayer, was capable of phagocytosing anti-Fc gamma RIII-coated erythrocytes. However, Fc gamma RIII on neutrophils, a molecule linked to the membrane by a phosphatidylinositol-glycan moiety, although binding anti-Fc gamma RIII-coated erythrocytes vigorously was incapable of mounting a phagocytic response. This deficiency correlates with the limited capacity of Fc gamma RIII on neutrophils to mediate superoxide generation and antibody-dependent cell-mediated cytotoxicity and it may be related to the unique structural features of Fc gamma RIII.
Cultures of peritoneal exudate cells rich in macrophages were studied for the secretion of lymphostimulatory molecules. Two conditions produced increased secretion: (a) addition to the cultures of various agents that readily interacted with macrophages, such as latex particles, antibody-coated red cells, endotoxin, Listeria organisms, or Be salt; or (b) addition of activated lymphocytes. In the first case the increased activity was found during the first 24 or 48 h after uptake of the stimuli. Increased activity was found in normal or peptone-stimulated macrophages but not in macrophages after injection of endotoxin or thioglycollate. The addition of T lymphocytes from Listeria-infected mice to macrophage cultures increased greatly the activities. This increase was also produced by addition to antigen-primed T cells together with antigen. The lymphocytes by themselves did not secrete active factors. The lymphostimulatory activities were tested on thymocyte DNA synthesis and on antibody formation in vitro. The latter assay was done on spleen cells from immunized mice where one striking effect was the stimulation of differentiation to antibody-secreting cells. Some dissociation of both activities (thymocyte DNA synthesis and B-cell differentiation) was observed with selected culture fluids.
Self-associating IgG rheumatoid factors isolated from three patients with rheumatoid arthritis activated human monocytes to release prostaglandin E2 as well as a previously described mononuclear cell factor, which stimulates human synovial cells to release additional prostaglandin E2 and collagenase. Thus, the immune complexes composed of self-associating IgG rheumatoid factors can contribute to the processes that destroy cartilage and bone in patients with rheumatoid arthritis.
The Fc receptors for IgG(FcγR) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of FcγR-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these FcγR in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monodonal antibodies specific for FcγR on human leukocytes has established the existence of three distinct FcγR and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of FcγR-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different FcγR is discussed. The results of these studies suggest that the ability of a given FcγR to trigger killing is sometimes dependent on the type of FcγR, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.
In spite of the physiologic and pathologic importance of tumor necrosis factor (TNF), the cellular factors that govern its release by macrophages (MΦ) are poorly understood, in comparison with other secretory products. We have studied the role of MΦ heterogeneity and of plasma membrane receptors in regulating TNF release in vitro. Resident and various exudate murine peritoneal MΦ populations were challenged with lipopolysaccharide (LPS) or different phagocytic particles, and TNF release assayed by cytotoxicity for L-929 fibroblasts. Resident peritoneal MΦ (RPMΦ) released a small amount of TNF in response to LPS whereas thioglycollate-elicited MΦ (TPMΦ) released high levels of TNF (5000 U/3 × 105 MΦ/ml). MΦ elicited by Bio-Gel polyacrylamide beads (BgPMΦ), another nonspecific inflammatory stimulus, or early in the course of intraperitoneal Bacillus Calmette-Guérin infection, before recruited cells become immunologically activated, released tenfold less TNF after the same stimulus. By contrast, TNF release in response to various phagocytic triggers was similar (∼300-600 U/3 × 105 MΦ/ml) in all MΦ populations including RPMΦ. The response by BgPMΦ to LPS was enhanced by pre-treatment in vitro with interferon-γ or thioglycollate broth. With respect to phagocytic receptor triggering we found that complement receptor type 3 (CR3) ligation or latex uptake did not mediate release of significant quantities of TNF (<48 U/3 × 105 MΦ/ml) by any MΦ, whereas ligation of the Fc receptor for IgG1/IgG2b subclasses or of receptors for zymosan particles sufficed, in the absence of ingestion. to induce release of circa 500 U/3 × 105 MΦ/ml TNF by all MΦ tested. Our studies show that MΦ vary in respect to priming for TNF release and that heterogeneity should be related to a particular triggering stimulus. Furthermore, the capacity of some MΦ populations to release unusually high levels of TNF depends on immune or nonspecific stimuli subsequent to the process of inflammatory recruitment.
Spleen cell populations depleted of both B and T lymphocytes produce interleukin 4 (IL-4) in response to stimulation with immunoglobulins bound to the surface of culture dishes. In the presence of interleukin 3 (IL-3), plate-bound (PB) IgE and PB-IgG1, IgG2a, and IgG2b are excellent stimulants, whereas PB-IgA and PB-IgM fail to stimulate IL-4 production. In the absence of IL-3, PB-IgE stimulates relatively modest production of IL-4, whereas PB-IgG2a generally does not. The response to PB-IgE is inhibited by soluble IgE; antibody to Fc gamma receptor II inhibits the response to PB-IgG2a. Thus, separate receptors mediate these stimulations, and Fc receptor cross-linkage is required for IL-4 production. Depletion of cells expressing asialo-GM1 does not diminish IL-4 production in response to PB immunoglobulins, indicating that natural killer cells are not essential for non-B, non-T cell production of IL-4. In addition to IL-4, non-B, non-T cells produce IL-3, but no detectable interleukin 2 or interferon gamma. Non-B, non-T cells may be an important source of lymphokines in a variety of immune responses and may serve to amplify the effects of T cells of the TH2 type.
The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.
Immune complexes (IC) are believed to play a role in the pathogenesis of some autoimmune diseases in which interleukin 1 (IL-1) and probably other cytokines also take part. This investigation shows that tetanus toxoid-human anti-tetanus toxoid IC induce human monocytes to release IL-1. The activity was identified as being mainly IL-1 beta by molecular size chromatography, isoelectric focusing, and anti-IL-1 beta affinity chromatography. Endotoxins were eliminated by repetitive washing of the IC suspension and by preincubation of IC with polymyxin B. The IL-1-inducing effect of IC was destroyed by heating at 80 degrees C, and it was not blocked by the cytoskeleton inhibitor cytochalasin B. IL-1 inhibitors were not detected in the supernatants.
The ability of immune complexes derived from patients with type II mixed essential cryoglobulinemia to trigger the production of interleukin 1 (IL 1) was investigated. Immune complexes containing either IgM/IgG or IgA/IgG aggregates were shown to induce IL 1 production in human peripheral blood monocyte-enriched populations. Interferon-gamma alone did not induce detectable IL 1, but increased the IL 1 production induced by the immune complexes. Tumor necrosis factor-alpha, which is a potent inducer of IL 1, also enhanced IL 1 production following stimulation with suboptimal doses of the immune complex. The findings suggest that immune complexes may induce inflammation partly due to their capacity to induce the synthesis of IL 1.
Cachectin (tumor necrosis factor) is a macrophage hormone strongly implicated in the pathogenesis of endotoxin-induced shock. The availability of a DNA probe complementary to the cachectin messenger RNA (mRNA), as well as a specific antibody capable of recognizing the cachectin gene product, has made it possible to analyze the regulation of cachectin gene expression under a variety of conditions. Thioglycollate-elicited peritoneal macrophages obtained from mice contain a pool of cachectin mRNA that is not expressed as protein. When the cells are stimulated with endotoxin, large quantity of additional cachectin mRNA is produced, and immunoreactive cachectin is secreted. Macrophages from mice of the C3H/HeJ strain do not produce cachectin in response to endotoxin. A dual defect appears to prevent cachectin expression. First, a diminished quantity of cachectin mRNA is expressed in response to low concentrations of endotoxin. Second, a post-transcriptional defect prevents the production of cachectin protein. Macrophages from endotoxin-sensitive mice do not produce cachectin if they are first treated with dexamethasone, apparently for similar reasons. These findings give new insight into the nature of the C3H/HeJ mutation and suggest an important mechanism by which glucocorticoids may act to suppress inflammation.
We have demonstrated herein that immune complexes (IC) bound to O+ human erythrocytes trigger the release of interleukin-1 (IL-1) from human monocytes in vitro. The efficiency of monocytes stimulation by erythrocyte-bound IC is in sharp contrast to the lack of stimulation of monocytes by soluble IC. The IL-1 activity triggered by erythrocyte-bound IC was lower than that induced by insoluble IC or opsonized fluorescent latex beads, but was greater than that induced by anti-D-treated O+ human erythrocytes. The magnitude of IL-1 release induced by insoluble IC and erythrocyte-bound IC were similar up to 18 hr of incubation. The IL-1 activity decreased in the supernatant of monocytes exposed to erythrocyte-bound IC for 40 hr, while the activity of IL-1 induced by insoluble IC remained at a high level. The physiological and physiopathological significance of triggering IL-1 release by erythrocyte-bound IC are discussed.
As a highly reactive substance produced in biological systems by the one-electron reduction of oxygen, superoxide (O(2) (-)) seemed a likely candidate as a bactericidal agent in leukocytes. The reduction of cytochrome c, a process in which O(2) (-) may serve as an electron donor, was found to occur when the cytochrome was incubated with leukocytes. O(2) (-) was identified as the agent responsible for the leukocyte-mediated reduction of cytochrome c by the demonstration that the reaction was abolished by superoxide dismutase, an enzyme that destroys O(2) (-), but not by boiled dismutase, albumin, or catalase. Leukocyte O(2) (-) production doubled in the presence of latex particles. The average rate of formation of O(2) (-) in the presence of these particles was 1.03 nmol/10(7) cells per 15 min. This rate, however, is only a lower limit of the true rate of O(2) (-) production, since any O(2) (-) which reacted with constituents other than cytochrome c would have gone undetected. Thus. O(2) (-) is made by leukocytes under circumstances which suggest that it may be involved in bacterial killing.