Chemiluminescence response of human phagocytes against IgG-opsonized staphylococci with and without protein A activities

The effects of immunological IgG binding to Staphylococcus aureus and IgG binding via protein A on the chemiluminescence (CL) response of human phagocytes were examined. The results obtained by enzyme immunoassay showed a clear correlation between the magnitude of the CL response and amount of IgG on protein A-deficient HL-87 strain. Despite no difference in protein A activity between 209P and Cowan I strains, the CL response to IgG-opsonized 209P cells was lower than that to Cowan I cells similarly opsonized. Moreover, the CL response to opsonized HL-87 cells was identical with that of opsonized Cowan I cells, which was a protein A-rich parent strain of the HL-87. The protein A activity of Cowan I cells was significantly decreased when the cells were treated with the Fc fraction of IgG before opsonization, but such a treatment did not change the phagocytic CL response. These results strongly suggest that IgG bound to protein A via its Fc portion has no effect on the phagocytic CL response and that IgG immunologically bound to S. aureus is responsible for the opsonization of the bacteria.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

Comparison of the interaction of tricyclic antidepressants with human polymorphonuclear leukocytes as monitored by the generation of chemiluminescence.

The interation of imipramine with human polymorphonuclear leukocytes (PMNs) results in a chemiluminescence (CL) response which has been attributed to the electronic excitation of the imipramine molecule resulting from a reaction of the drug with reactive oxygen species. In order to determine what portion of the tricyclic molecule is involved in this reaction, the interaction of other tricyclics with PMNs was monitored by chemiluminescence. It was observed that tricyclic antidepressants having a carbon atom at position 5 of the ring moiety (amitriptyline, for example) did not yield CL with either resting or zymosan-activated PMNs. In fact this group of compounds inhibited the zymosan-induced CL response. However, CL was observed, with both resting and metabolically-activated PMNs, from several tricyclics having a heterocyclic nitrogen at position 5. These included imipramine, desipramine, opipramol and iprindole. Chlorimipramine, which has a chlorine atom at position 3 of the ring system, failed to yield CL with resting or stimulated cells. Similarly, imipramine N-oxide failed to yield CL with resting cells, but enhanced CL was observed with zymosan-activated PMNs. On the basis of these observations it appears that some aspect of the ring moiety, other than just a heterocyclic nitrogen, facilitates a reaction between these molecules and reactive oxygen which culminates in the generation of CL.     

Three-peaked chemilluminescent response of human peripheral blood leukocytes following stimulation with phytohaemagglutinin (PHA)

Luminol-enhanced chemiluminescence (CL) was used to examine the response of various leukocyte populations following stimulation with a crude extract of Phaseolus vulgaris, namely phytohaemagglutinin (PHA-C). Populations stimulated included a human peripheral mixed leukocyte preparation (MLP), and purified preparations of lymphocytes, monocytes and polymorphonuclear leukocytes (PMNL). Mouse peritoneal exudate cells and the lymphocytic cells lines Molt #4 and Daudi were also stimulated. Following stimulation, a characteristic three-peaked chemiluminescent response was obtained from the MLP population. Little or no response was obtained from the purified lymphocytes. Monocytes produced a sharp peak corresponding to the second peak of the MLP response and PMNL produced a broad peak corresponding to the third peak of the MLP response. Mouse peritoneal exudate cells containing lymphocytes and monocytes/macrophages showed a two-peaked stimulation which corresponded to the first two peaks of the MLP response. Molt #4 and Daudi showed no chemiluminescence if stimulated individually, but if added to a MLP substantial enhancement of the first and second peaks was observed. These results indicate some form of lymphocyte/monocyte interaction leading to enhanced CL following PHA-C stimulation.     

Low responsiveness of human neonatal lymphocytes to a B-cell mitogen, Staphylococcus aureus Cowan I (SpA CoI)

Responses of neonatal and adult lymphocytes to various mitogens were studied. Lymphocytes from umbilical cord blood (UCB) responded well to both phytohemagglutinin and concanavalin A, and also to pokeweed mitogen and Staphylococcus aureus Protein A. The responses of UCB lymphocytes to these mitogens were not significantly lower than those of adult peripheral blood lymphocytes (PBL). In contrast, UCB lymphocytes showed only a minimal response to killed Staphylococcus aureus Cowan I (SpA CoI), a potent B-cell mitogen for human PBL, although the proportion of B cells in UCB was not less than that in PBL. The low level of response of lymphocytes from UCB to SpA CoI was not ascribed to differences in dose response or kinetics. Purified B cells from UCB were not stimulated by SpA CoI either, suggesting tht the low responsiveness was not due to the suppressive effect of T cells or macrophages, but to some intrinsic defect in B cells in UCB. These results suggest that the B cells in neonates may be more immature than the T cells.     

Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 μg/ml)-pretreated PMNs than in non-pretreated ones. The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.     

Response of human B cells to Staphylococcus aureus Cowan I: T-independent proliferation and T-dependent differentiation to immunoglobulin secretion involve subsets separable by rosetting with mouse erythrocytes

We separated T-depleted mononuclear cells into subsets by rosetting with mouse erythrocytes and studied proliferation and differentiation responses to Staphylococcus aureus Cowan I (SAC), pokeweed mitogen (PWM), and a combination of the two polyclonal activators. All of the T cell-independent proliferation of unfractionated B cells in response to SAC was attributable to mouse erythrocyte rosette-forming cells (BMR+). BMR- cells were not stimulated to proliferate by SAC in the presence or absence of T cells, but did proliferate to PWM plus irradiated T cells. Co-stimulation of BMR+ cells with SAC and PWM in the presence of autologous T cells did not lead to immunoglobulin secretion. The B cells stimulated to divide by SAC apparently do not become responsive to B cell differentiation factors and are distinct from those that undergo T cell-dependent differentiation.     

Induction of nonspecific, proliferative dependent suppressor T lymphocytes in human mixed lymphocyte cultures

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), or Staphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA CoI stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.     

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.     

Induction and suppression of immunoglobulin synthesis in cultures of human lymphocytes: effects of pokeweed mitogen and Staphylococcus aureus Cowan I

The synthesis and secretion of immunoglobulins by human peripheral blood mononuclear cells in cultures stimulated with pokeweed mitogen or Staphylococcus aureus Cowan I were evaluated by enumeration of cells containing cytoplasmic immunoglobulins and cells actively secreting immunoglobulins, and by quantitation of immunoglobulins released into culture supernatants. The two mitogens caused comparable stimulation of immunoglobulin production; however, in contrast to pokeweed mitogen, S. aureus was active in cultures depleted of T lymphocytes, and its stimulatory effects were resistant to the influence of suppressor T cells generated by co-stimulation with concanavalin A or by preincubation without mitogenic stimulus. These results indicate distinct pathways of induction and suppression of immunoglobulin synthesis for these two polyclonal B cell activators, and suggest that stimulation by S. aureus is less thymus dependent than that induced by pokeweed mitogen.     

Stimulation of Immunoglobulin Production from Human B Lymphocytes by Staphylococcus aureus: Effects of Monocytes and Con A-Induced Suppressor Cells

Significant immunoglobulin (Ig) production by human peripheral blood lymphocytes was induced in vitro by stimulating the cells with pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SpA CoI). IgG, IgM, and IgA were determined by a combination of the latex fixation test and radioimmunoassay. High levels (1,000 to 5,000 μg/ml) of IgG and IgM and a lesser amount of IgA were constantly produced during 7 to 8 days of incubation with both stimulants. Ig production induced by SpA Col stimulation was independent of the presence of T cells, while Ig production induced by PWM required T cells exclusively. Depletion of monocytes in the culture caused but a slight decrease in Ig production (particularly in the case of IgG). While the addition of a small number of monocytes enhanced IgG induction by both stimulants, coculture with an excess number of monocytes inhibited Ig induction (particularly IgG) by PWM stimulation but not by SpA CoI stimulation. Marked suppression of Ig production (IgG, IgM, and IgA) was observed in cocultures with Con A-activated T cells. The phenomena of suppression were observed in both the SpA Col-stimulated and PWM-stimulated lymphocytes. These data indicate that Ig production from B cells stimulated with a polyclonal B cell activator, SpA CoI, was independent of T cells and relatively of independent of monocytes, but could be subjected to the regulation of the Con A-induced suppressor T cells.     

Intracellular localization of Staphylococcus aureus within primary cultured mouse kidney cells.

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among these of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.     

Distinction of two subtypes of human leukocyte interferon (IFN-alpha) on B cell activation. B cell proliferation by two subtypes of IFN-alpha

We examined the effect of interferon (IFN), with particular emphasis on the effects of the two subtypes of IFN-alpha (IFN-alpha A and IFN-alpha B) on the B cell proliferation induced by Staphylococcus aureus Cowan I bacterium (SpA Col). An increase of SpA Col-induced proliferation was observed in the presence of 100 to 1000 U/ml of IFN-alpha, but a decrease of SpA Col-induced proliferation was observed in the presence of 1000 to 10,000 U/ml of IFN-beta. The two subtypes of IFN-alpha had different effects on cell proliferation; a significant enhancement was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha A, but inhibition was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha B. In the reconstitution test of the two subtypes of IFN-alpha, the boundary between enhancement and inhibition of SpA Col-induced proliferation was revealed when the proportion of IFN-alpha A and IFN-alpha B (IFN-alpha A:IFN-alpha B) ranged between 8:2 and 9:1. Toward the SpA Col-induced responses, the above IFN were all found to act on B cells directly, independent of the presence of T cells. Proliferative responses by IFN-alpha and IFN-alpha A, however, were shown to be slightly dependent on the presence of monocytes. The lymphocyte proliferation induced by other mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and protein A of S. aureus) were all inhibited by the above IFN.     

Induction of human immunoglobulin secretion. II. T lymphocyte dependency and radiosensitivity of T-cell help for induction of B-cell differentiation by Staphylococcus aureus strain Cowan I

The induction of peripheral blood B lymphocytes to mature to immunoglobulin-secreting cells (ISC) when stimulated by Staphylococcus aureus strain Cowan I was found to be T helper cell-dependent (J. Immunol.127, 1044, 1981). The nature of T help was studied in B- and T-cell separation and reconstitution experiments. T helper cells for Cowan I were very radiosensitive (D₃₇ < 500 rad) in comparison to helpers for pokeweed mitogen (PWM) (D₃₇ > 2000 rad). PWM synergized with Cowan I in induction of ISC, and helper T cells for dual stimulation were also radioresistant. The ratio of T to B cells was found to be critical in judging reactivity of donors for both PWM and Cowan I. T cells stimulated with PWM, but not Cowan I, produced T cell-replacing factors essential for Cowan I-induced maturation of B cells. Irradiation of T cells prior to PWM stimulation increased the level of such helper factors. Poor responders to Cowan I, as judged by mononuclear cell cultures, had apparently few helpers for the bacterial stimulant, compared to high responders. Cowan I helper T-cell activity did not appear to be due to protein A leaking from the bacteria and stimulating T cells. In all these experiments, induction of ISC by Cowan I was completely dependent on T cells or factor, providing a good model for investigation of B-cell differentiation regulated by a unique subset of radiosensitive T helper cells.     

Cell-Dependent Chemiluminescence. Modulation of the N-Formyl Chemotactic Peptide (Fnlpntl) Mediated Oxidative Burst in Human Polymorphonuclear Leukocytes (Pmnl) By Murine Monoclonal Antibody Nms-1

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL.

We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL ± moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition.

Stimulation of human PMNLs by 10nM FNLPNTL induced a rapid burst of chemiluminescence which peaked ～5min after stimulus addition. The subsequent addition of moAB NMS-1 (?2μg/ml DPBS(+)—0.1% HSA, 37°C) to FNLPNTL-stimulated PMNLs—after the FNLPNTL-mediated response had already decayed (16-18 min) – without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent.

Treatment of PMNLs with moAB NMS-1 (? 1μg/ml DPBS(+)—0.1% HSA, 3 min, 37°C)—prior to FNLPNTL (10nM) stimulation – increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak ～16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the respone was not changed compared to controls which received no moAB NMS-1 treatment.

The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (5–60 min), persistent activation was not detected.     

Increased native chemiluminescence in granulocytes isolated from synovial fluid and peripheral blood of patients with rheumatoid arthritis

Polymorphonuclear leukocytes (PMNs) isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis and from peripheral blood of volunteers were stimulated with 12-Phorbol-13-myristate acetate (PMA). No significant differences in luminol-amplified chemiluminescence were found between different patients and control groups. However, two distinct patterns of native chemiluminescence were observed. Type I showed no, or only a small, increase in native chemiluminescence with integral counts over 30 min less than 3 × 10⁵ cpm, and the majority of samples from volunteers were of this type. Type II was characterized by a burst of native chemiluminescence starting 8 to 15 min after cell stimulation. It was found in most PMN samples from patients with rheumatoid arthritis. Integral counts over 30 min were always higher than 10⁶ cpm and as high as 10⁸ cpm in some cases. A strong inhibition of the Type II native chemiluminescence was caused by desferal, catalase, thiourea, and glutathione. However, the luminol-amplified chemiluminescence remained unchanged or was only slightly decreased under the same experimental conditions. Sodium azide strongly inhibited both kinds of luminscence. Hydroxyl radicals, formed in a Fenton reaction, may be important intermediates in the Type II native chemiluminescence.     

Rapid accumulation of polymorphonuclear neutrophils in the Corpus luteum during prostaglandin F(2α)-induced luteolysis in the cow

Prostaglandin F(2α) (PGF(2α)) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF(2α) administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF(2α) injection. The number of PMNs was increased at 5 min after PGF(2α) administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF(2α) directly stimulated P-selectin protein expression at 5-30 min in luteal endothelial cells (LECs). Moreover, PGF(2α) enhanced PMN adhesion to LECs, and this enhancement by PGF(2α) was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF(2α) is crucial in PMN migration. In conclusion, PGF(2α) rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF(2α) may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.     

The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection.

We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.     

The effect of bombesin-related peptides on the phagocytic function of mouse phagocytes in vitro

Bombesin (BBS) at doses of 0.1, 1.0, 10.0 and 100.0 nM stimulated chemiluminescence (CL) production by phagocytic cells (monocytes, macrophages and polymorphonuclear leucocytes) in mice in the presence of ZAP (opsonized zymosan particles containing luminol). These data suggest that BBS increased the phagocytic function of mouse phagocytes. BBS-related peptides, gastrin-releasing peptides (GRP)-27, GRP-14, GRP-10 and neuromedin B, also induced similar CL responses compared with BBS. The CL response elicited by BBS was depressed dramatically by various concentrations of EGTA (a Ca++ chelator), indicating that a Ca++ pathway may play a key role in the BBS-stimulated CL response.