Cytosolic free calcium is essential for immunoglobulin G-stimulated intracellular killing of Staphylococcus aureus by human monocytes.

Earlier studies have shown that the intracellular killing of Staphylococcus aureus by human monocytes requires continuous stimulation by serum factors, e.g., immunoglobulin G (IgG). In the present study, we demonstrate that IgG, at concentrations that stimulate the intracellular killing of S. aureus, induces a transient increase in the intracellular free calcium concentration ([Ca2+]i) in monocytes. The Ca2+ ionophores A23187 and ionomycin stimulate the killing process as efficiently as IgG does and initiate O2- production in resting monocytes but not in monocytes containing bacteria. The Ca2+ ionophore-stimulated killing process was markedly inhibited by the NADPH oxidase inhibitor diphenyleneiodonium bisulfate, which indicates that these ionophores stimulate oxygen-dependent bactericidal mechanisms. Reduction of the [Ca2+]i to values below 1 nM, obtained by loading monocytes with MAPT/AM (1,2-bis-5-methyl-aminophenoxylethane-N,N,N',N'-tetraacetoxymet hyl acetate) in the absence of extracellular Ca2+, rendered the cells unresponsive to IgG or Ca2+ ionophore stimulation of the intracellular killing of S. aureus, but the response could be restored by reincubating these cells in the presence of extracellular Ca2+. It is concluded that cytosolic free Ca2+ is essential for the IgG-stimulated intracellular killing of S. aureus by human monocytes.     

Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes.     

Bruton's tyrosine kinase is not essential for LPS-induced activation of human monocytes

BACKGROUND: X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The natural disease model, the X-linked immunodeficiency mouse, shows a less severe phenotype, indicating a different requirement of Btk in human and mouse B cells. Btk is also expressed in the myeloid line and participates in LPS signaling. Deficient oxidative burst and myeloid differentiation have been reported in the X-linked immunodeficiency mouse, but the precise mechanism and relevance of Btk activity in human monocytes is poorly understood. OBJECTIVE: The apparent absence in XLA of clinical manifestations of myeloid deficiency prompted us to explore the relevance of complete Btk absence in human myeloid cells. METHODS: Seven patients with XLA with BTK mutations conditioning a null protein expression were included in the study. Monocyte LPS-induced mitogen-activated protein kinase activation, TNF-alpha and IL-6 production in monocytes, and oxidative burst in monocytes and granulocytes were analyzed by means of flow cytometry. RESULTS: We show that in response to LPS, Btk-null monocytes from patients with XLA induce early mitogen-activated protein kinase activation and intracellular TNF-alpha and IL-6 production with the same intensity as cells from age- and sex-matched control subjects. In addition, the oxidative burst in response to LPS and other stimulants was completely normal in Btk-null monocytes and neutrophils. CONCLUSION: Our results indicate that Btk is not essential for early LPS signaling in human monocytes and that different Btk dependency might exist between human and mouse myeloid cells. CLINICAL IMPLICATIONS: These findings provide a better understanding of XLA, and they show the differences between human XLA and murine Xid models.     

Effect of probenecid on phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes.

The present study concerns the effects of probenecid on the phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes. In both monocytes and granulocytes the inhibitory effect on phagocytosis was very small. Inhibition of intracellular killing of S. aureus by monocytes and granulocytes by probenecid was concentration dependent, being half-maximal at about 2 mM probenecid, and near-maximal at about 5 mM probenecid. The intracellular killing could also be inhibited when probenecid was added when this process was already started. Probenecid also inhibited the intracellular killing of E. coli by granulocytes, but not by monocytes. In the concentration range used, probenecid had no toxic effect on phagocytes or bacteria during the 2 hr of the experiments.     

Effect of concanavalin A on intracellular killing of Staphylococcus aureus by human phagocytes.

This study concerns the influence of concanavalin A (Con A) on phagocytosis and intracellular killing of Staphylococcus aureus by human monocytes and granulocytes. Con A binds to S. aureus, monocytes, and granulocytes, and is not opsonic. Con A stimulates the killing of intracellular serum opsonized S. aureus by monocytes, but not by granulocytes. This stimulation of intracellular killing was inhibited by alpha-methyl-mannoside, indicating that the process occurs via Con A specific membrane binding sites. Unlike (tetravalent) Con A, divalent succinyl-Con A does not stimulate intracellular killing, indicating that the lectin valency is important for this stimulation. Con A bound to Sephadex particles, that can not be ingested by monocytes, does not stimulate intracellular killing of S. aureus either, although it, like free Con A, stimulates H2O2 production. Pre-incubation of monocytes with Con A inhibited Fc gamma and C3b-mediated ingestion of S. aureus as well as stimulation of the killing by serum. Divalent Con A had no effect on these functions. This inhibition by Con A is in all probability due to a steric impedance of Con A with respect to the interaction of IgG and C3b with their membrane receptors. Fluorescence techniques showed that Con A was localized on the membrane and in the cytoplasm of the monocytes, whereas granulocytes had only membrane bound lectin. Taken together, these findings suggest that cell penetration by the lectin is obligatory for the stimulation of intracellular killing.     

Kinetics of intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes

The intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes was investigated independently of the ingestion of these bacteria. Granulocytes were allowed to phagocytose preopsonized bacteria for only 3 min, after which the noningested bacteria were removed by differential centrifugation and 2 washes. With this technique, the number of viable cell-associated bacteria at the start of the assay, determined after lysis of the granulocytes, includes about 80% intracellular bacteria. Intracellular killing depends on (a) the temperature (no killing occurring at 4°C, maximal killing at 33–39 °C, and a decrease in the capacity of the granulocytes to kill ingested bacteria at temperatures above 42 °C), and (b) the number of bacteria ingested (after phagocytosis at bacteria-to-cell ratios of 100 : 1 and 1000 : 1, not all of the ingested bacteria are killed, whereas after phagocytosis at lower bacteria-to-cell ratios, almost all ingested bacteria are killed). To determine the maximum number of bacteria that can be killed by granulocytes, intracellular killing was measured after phagocytosis of bacteria at various bacteria-to-granulocyte ratios in the presence of phenylbutazone, a drug which inhibits killing during the ingestion period. Phenyl-butazone proved to be a useful tool in the study of intracellular killing, since this drug provides a reversible inhibition of the killing when granulocytes are incubated in its presence for up to 3 min, whereas after longer incubation, the inhibitory effect is irreversible. Calculation based on the data obtained in this study gave maximum rates of intracellular killing amounting to 3.7 × 10⁵ bacteria/5 × 10⁶ granulocytes/min for Staph. aureus and 8.5 × 10⁵ bacteria/5 × 10⁶ granulocytes/min for E. coli. Using the rate of intracellular killing after phagocytosis at a bacteria-to-granulocyte ratio of 1 : 1 and the rate of ingestion obtained in an earlier study, we were able to compute the theoretical numbers of viable extracellular not (yet) ingested, viable intracellular, killed intracellular and total intracellular bacteria. The theoretical curves fit well with the experimental data.     

Protein tyrosine kinase regulates FAS-mediated apoptosis in human BCG-infected monocytes.

Apoptosis of monocytes/macrophages has emerged as a central regulatory event in the defense against mycobacterial infections. The involvement of protein tyrosine kinases (PTK) in Fas-mediated apoptosis in T cells is well established, but the possible role of PTK in Fas-dependent death of human bacillus Calmette-Guerin (BCG)-infected monocytes remains unclear. Here, we first examined the expression and function of Fas on BCG-infected human monocytes by flow cytometry. The results demonstrated that BCG-infected monocytes expressed significant Fas protein levels. In addition, engagement of the Fas antigen with its agonistic antibody (Ab) resulted in apoptosis of monocytes, as monitored by DNA analysis and fluorescence-activated cell sorter (FACS) analysis. The apoptotic action of Fas was suppressed significantly by genistein, indicating a role for PTK in this death process. Consistent with this observation, herbimycin A and tyrphostin, two selective tyrosine kinase inhibitors with different mechanisms of action, effectively inhibited Fas-mediated apoptosis of BCG-infected monocytes, as demonstrated by DNA content analysis. Moreover, we confirmed the effect of genistein, herbimycin A, and tyrphostin by examining apoptosis with the terminal transferase dUTP nick endlabeling (TUNEL) assay. Collectively, these data demonstrate that Fas-induced apoptosis may represent an important mechanism for eliminating BCG-activated human monocytes and that this apoptosis is due, at least in part, to signaling via a PTK pathway.     

Stimulation of the intracellular killing of Staphylococcus aureus by human monocytes mediated by Fcγ receptors I and II

Previous studies have shown that intracellular killing of bacteria by monocytes is stimulated by interaction between IgG and Fey receptors (FcγR) in the membrane of these cells. In the present study anti-FcγR monoclonal antibodies (mAb) were used to investigate the relative contributions of the various classes of FcγR to the intracellular killing of Staphylococcus aureus by human monocytes and the biochemical pathways involved. Anti-FcγRI or anti-FcγRII mAb, but not anti-FcγRIII mAb, efficiently stimulated the intracellular killing of bacteria by monocytes. Cross-linking FcγRI or FcγRII, but not FcγRIII, on monocytes with mouse anti-FcγR mAb followed by bridging with F(ab′)₂ fragments of goat anti-mouse IgG enhanced this process. Since the NADPH oxidase inhibitor diphenyleneiodonium blocked the FcγR-mediated intracellular killing of S. aureus, oxygen-dependent bactericidal mechanisms are most probably involved. Cross-linking FcγRI or FcγRII but not binding of the mAb to the FcγR on monocytes activated phospholipase C, as demonstrated by the increase in the intracellular concentration of inositol- (1,4,5)-triphosphate. The enhanced intracellular killing stimulated by cross-linking FcγR on monocytes was completely blocked by U-73122, an inhibitor of phospholipase C-dependent processes. Protein kinase C activity, but not the rise in the cytosolic free Ca⁺⁺ concentration or pertussis toxin-sensitive G proteins, is essential for the FcγR-mediated intracellular killing of bacteria by monocytes. Together, these results demonstrate that cross-linking FcγRI or FcγRII is equally effective in stimulating the intracellular killing of bacteria by monocytes and that this stimulation is a phospholipase C-dependent process.     

Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes.

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG (2-10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgG1 and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.     

Phagocytosis and intracellular killing of Staphylococcus aureus by normal mouse peritoneal macrophages.

Although Staphylococcus aureus is incapable of intracellular multiplication in cultured mouse peritoneal macrophages, it is killed at a much slower rate than the avirulent Staphylococcus epidermidis. In addition to the presence of capsular material which inhibits phagocytosis of specific strains of S. aureus, the data show that a number of cellular and environmental factors affect the functional capacities of mononuclear phagocytic cells. The data obtained by varying the initial level of infection indicate that the number of ingested bacteria may subsequently alter the kinetics of intracellular killing. In vitro maturation of macrophages in culture was also found to exert a pronounced effect on the kinetics of bacterial death.     

Selective killing of human monocytes and cytokine release provoked by sphingomyelinase (beta-toxin) of Staphylococcus aureus.

The best-known activity of Staphylococcus aureus sphingomyelinase C, alias beta-toxin, is as a hemolysin that provokes hot-cold lysis of erythrocytes which contain substantial amounts of sphingomyelin in the plasma membrane. Sheep erythrocytes are most susceptible, and we found that one hemolytic unit, representing the toxin concentration that elicits 50% hemolysis of 2.5 X 10(8) erythrocytes per ml, corresponds to 0.05 enzyme units or to approximately 0.25 microg of sphingomyelinase per ml. The cytotoxic action of beta-toxin on nucleated cells has not been described in any detail before, and the present investigation was undertaken to fill this information gap. We now identify beta-toxin as a remarkably potent monocytocidal agent. At a concentration of 0.001 U/ml, corresponding to approximately 5 ng/ml, beta-toxin killed over 50% of human monocytes (10(6) cells per ml) within 60 min. By contrast, 1 to 5 microg of beta-toxin per ml had no cytocidal effects on human granulocytes, fibroblasts, lymphocytes, or erythrocytes. A selective monocytocidal action was also observed with sphingomyelinase C from Bacillus cereus and a Streptomyces sp., whereas phospholipase A2 and phospholipase D at 100 U/ml were without effect. Monocytes succumbing to the action of beta-toxin processed and released interleukin-1beta, soluble interleukin-6 receptor, and soluble CD14 into the supernatant. Thus, monocyte killing by beta-toxin is associated with cytokine-related events that are important for the initiation and progression of infectious disease. These findings uncover a potentially important role for sphingomyelinase as a determinant of microbial pathogenicity.     

Respiratory activity is essential for post-exponential-phase production of type 5 capsular polysaccharide by Staphylococcus aureus.

Capsule formation is believed to have a significant role in bacterial virulence. To examine the possible involvement of capsular polysaccharide (CP) from Staphylococcus aureus in the pathological mechanisms associated with staphylococcal infections, we investigated the influence of respiratory activity on type 5 CP production by S. aureus grown in the presence of various concentrations of dissolved oxygen or nitrate. The effects of several metabolic inhibitors (arsenite, cyanide, azide, trimethylamine N-oxide, 2-heptyl-4-hydroxyquinoline N-oxide, and 2,4-dinitrophenol) were also tested. The metabolism of the bacteria was estimated by measuring their reductive capacity and by monitoring the pH and concentrations of fermentation products. Type 5 CP was always produced by S. aureus during the exponential phase of growth under all culture conditions tested. In contrast, post-exponential-phase CP production appeared to be strictly dependent on the respiratory activity. Since post-exponential-phase CP production contributes at least two-thirds of the total CP obtained, the influence of S. aureus respiration on CP production might be of some importance in the process of infection.     

Phagocytic killing of encapsulated and microencapsulated Staphylococcus aureus by human polymorphonuclear leukocytes.

Phagocytosis by human polymorphonuclear leukocytes (PMNs) is an important host defense against infections caused by Staphylococcus aureus. Using an in vitro assay, we compared the opsonic requirements for phagocytic killing of prototype strains of encapsulated (type 1) and microencapsulated (type 5 and type 8) S. aureus by human PMNs. More than 85% of broth-grown, logarithmic-phase type 5 and 8 S. aureus organisms were killed by PMNs incubated with fresh normal human, rabbit, or guinea pig serum with complement activity. Under similar conditions, the highly encapsulated type 1 strain was not killed. Both encapsulated and microencapsulated strains were opsonized for phagocytosis by heat-inactivated serum raised in rabbits to killed bacteria. Opsonization by homologous serum was required for phagocytosis of the type 1 strain. In contrast, microencapsulated type 5 and 8 S. aureus organisms were killed by heat-inactivated rabbit serum raised to type 5, type 8, or nonencapsulated isolates; this result suggested that antibodies to the capsule or to cell wall components other than the capsule could opsonize these organisms for phagocytosis. The specificity of the assay was confirmed with capsule type 5-specific monoclonal antibodies, which were opsonic only for the type 5 S. aureus isolate. These studies indicate that, unlike the highly encapsulated type 1 strain, broth-grown microencapsulated S. aureus strains do not resist opsonophagocytic killing in vitro by normal serum.     

Fc gamma receptor recognition of IgG ligand by human monocytes and macrophages

We examined the binding characteristics of human monocytes and macrophages with the IgG ligands, human monomeric IgG and a small human IgG aggregate, trimeric IgG. Our purpose was to utilize fresh monocytes, in vitro cultured monocytes, and alveolar macrophages in direct and indirect binding experiments. Freshly isolated monocytes expressed only a single binding site for IgG monomer and IgG trimer. In contrast, in vitro cultured monocytes, gamma-interferon-treated monocytes, and freshly isolated alveolar macrophages expressed a single binding site for IgG monomer and, in addition, a high and low affinity binding site for IgG trimer. The high affinity binding site for IgG trimer (Kd approximately equal to 1 nM) appeared identical to the binding site for IgG monomer. The low affinity binding site for IgG trimer (Kd = 50 to 250 nM) appeared to be due to Fc gamma RII, because antibody to Fc gamma RII inhibited its expression. Since Fc gamma RII, in contrast to Fc gamma RI, does not bind monomeric IgG, the data suggest that this low affinity receptor for trimeric IgG, Fc gamma RII, can bind low molecular weight circulating immune complexes at concentrations 10- to 100-fold lower than Fc gamma RI. Thus, these studies suggest that at 37 degrees C, macrophage Fc gamma RII may play a functional role in the recognition of small molecular weight immune complexes.     

Participation of immunoglobulins and complement components in the intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes.

Immunoglobulins and complement components are required for optimal ingestion and optimal killing of microorganisms by granulocytes. The degree of opsonization of microorganisms necessary for their ingestion was lower than that required for the killing of these bacteria during the ingestion phase. Killing during this phase was found to depend mainly on the presence of heat-labile opsonins, probably C3b, present on the microorganisms. Extracellular immunoglobulin G (IgG) and C3b were indispensable for optimal intracellular killing after ingestion was complete. This was established with an assay permitting assessment of the course of the number of viable intracellular bacteria independent of the ingestion of new live bacteria. Maximal intracellular killing by human granulocytes of ingested catalase-positive (Staphylococcus aureus and Escherichia coli) or catalase-negative (Streptococcus pyogenes and S. pneumoniae) microorganisms was found only when fresh serum was present extracellularly. Killing was suboptimal in the absence of serum. With heat-inactivated serum, the killing index lay between the indices obtained in the presence and absence of fresh serum. The stimulatory activity of heat-inactivated serum was most probably due to the interaction of IgG with the Fc receptor on the granulocyte membrane, since IgG subclasses IgG1 and IgG3 as well as pFc fragments of IgG stimulated the intracellular killing to the same degree as heat-inactivated serum did. In addition, (Fab1)2 fragments of IgG did not stimulate killing, and reduced killing was observed in the presence of heat-inactivated serum after reduction of the number of Fc receptors. The extra stimulation of the killing process in the presence of fresh serum compared with heat-inactivated serum was due to the interaction between membrane receptors and complement--most probably C3b generated by both the classical and the alternative pathways of complement activation. This conclusion is based on results obtained with sera in which one or both complement pathways were blocked, on the restoration of the killing-stimulatory activity of C3-deficient serum after addition of fresh C3, and on the reduced killing observed in the presence of fresh serum after reduction of the number of C3 receptors by the use of pronase or antigranulocyte serum.     

Trp-Lys-Tyr-Met-Val-D-Met stimulates superoxide generation and killing of Staphylococcus aureus via phospholipase D activation in human monocytes

Among the phagocytic leukocytes, monocytes have the important role of clearing out parasitic microorganisms. They accomplish this through production of toxic metabolites of oxygen. Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), a peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes, including monocytes, binds to a unique cell surface receptor and stimulates superoxide generation, killing of Staphylococcus aureus, and activation of phospholipase D (PLD) in human monocytes. Preincubation of the cells with a PI-specific phospholipase C (PLC) inhibitor (U-73122), protein kinase C inhibitor (GF109203X), or intracellular Ca2+ chelator (BAPTA/AM) before the peptide stimulus totally inhibits the peptide-induced PLD activation and superoxide generation. On the other hand, tyrosine kinase inhibitor genistein only partially inhibits the peptide-induced processes. The peptide-induced bacteria killing activity shares regulatory mechanisms for PLD activation with the superoxide generation, which is inhibited in the presence of 1-butanol. We suggest that the peptide stimulates PLD downstream of PLC activation and PLD activation in turn is essential for the peptide-induced immunological functions such as the superoxide generation and killing of bacteria by human monocytes.     

Pertussis toxin partially inhibits phagocytosis of immunoglobulin G-opsonized Staphylococcus aureus by human granulocytes but does not affect intracellular killing.

The aim of the present study was to determine whether pertussis toxin (PT)-sensitive GTP-binding proteins (G proteins) are involved in the signal transduction pathway(s) used for phagocytosis and intracellular killing of bacteria by human granulocytes. Treatment of granulocytes with PT resulted in decreased phagocytosis of immunoglobulin G (IgG)-opsonized Staphylococcus aureus but did not affect subsequent intracellular killing of these bacteria. PT also caused a decrease in the extracellular release of superoxide anion (O2-) and hydrogen peroxide (H2O2) by granulocytes in response to S. aureus opsonized by IgG. However, neither the phagocytosis nor the intracellular killing of S. aureus opsonized by fresh serum was affected by PT, and the release of O2- was partially inhibited. The release of O2- in response to serum-treated zymosan, opsonized mainly by complement components, was also only partially inhibited by PT. It is therefore possible that PT inhibits responses mediated through complement receptors to a lesser extent than those mediated via Fc gamma receptors. The results of this study indicate that PT-sensitive G proteins are involved in the signal transduction pathways that mediate the phagocytosis of IgG-opsonized bacteria and the accompanying respiratory burst.     

Age-dependent alterations of Fc gamma receptor-mediated effector functions of human polymorphonuclear leucocytes.

Changes in the effector functions in polymorphonuclear leucocytes (PMNL), harvested from blood of young and aged healthy subjects of both sexes, were studied. FC gamma-receptor (Fc gamma R)-mediated incorporation of IgG coated 51Cr-HRBC significantly increased in the aged male group, while the phagocytosis of pre-opsonized fungi (Saccharomyces cerevisiae and Candida albicans) was independent of both the age and sex. However, the intracellular killing capacity of neutrophils obtained from aged male subjects significantly decreased toward 51Cr-labelled c. albicans. The antibody-dependent cellular cytotoxicity (ADCC) was also impaired with ageing in both sexes. The age-dependent decrease in the effector functions of PMNL may be explained, among others, by the fact that during yeast cell incorporation the increased cAMP level does not return to the basic level in the old group. On the other hand, the cGMP level which increased in PMNL of aged subjects does not show any progressive increase as in the young subjects, but remains unchanged. The oxidative metabolism producing free radicals being necessary for the effective intracellular killing and ADCC diminished in PMNL of aged subjects of both sexes. The above findings indicate that the adaptation of cyclic nucleotide system and the oxidative burst to the cell activation becomes impaired with ageing.     

Synovial fluid inhibits killing of Staphylococcus aureus by neutrophils.

Serum in the extracellular environment promotes neutrophil bactericidal activity apart from its opsonizing properties. We examined the effect of non-inflammatory osteoarthritic synovial fluid on serum-mediated neutrophil killing of Staphylococcus aureus. This was done to evaluate the effect of synovial fluid on neutrophil bactericidal activity independent of opsonin concentration. With an initial inoculum of 5 X 10(6) CFU/ml, 1.47 +/- 0.14% bacteria survived after 120 min of incubation with 10% serum and neutrophils. In contrast, 4.07 +/- 0.33% bacteria survived after incubation in serum plus synovial fluid (P less than 0.001). This inhibitory effect was directly related to the concentration of synovial fluid in the incubation mixture. Increasing the concentration of synovial fluid resulted in an increased percent survival. Studies utilizing preopsonized bacteria and radiolabeled organisms demonstrated that synovial fluid did not interfere with opsonization or phagocytosis. Intracellular bactericidal activity was assayed separately from phagocytosis by utilizing a brief ingestion period followed by the removal of extracellular bacteria by either differential centrifugation or lysostaphin treatment. The reincubation of cells and associated bacteria with serum or serum plus synovial fluid revealed that synovial fluid significantly inhibited the promoting effect of serum on neutrophil bactericidal activity. After 60 min of incubation with 10% serum, 13.0 +/- 1.2% bacteria survived, whereas 21.5 +/- 2.3% survived after incubation in serum plus synovial fluid (P less than 0.005). Superoxide production was not affected by the presence of synovial fluid. These findings suggest that the inhibitory effect of synovial fluid is due to an interaction between synovial fluid and the serum factors that promote intracellular killing.     

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.