Effect of piliation on interactions of Haemophilus influenzae type b with human polymorphonuclear leukocytes.

Piliated, adherent (P+) and nonpiliated, nonadherent (P-) strains of Haemophilus influenzae type b (Hib) were compared with respect to their ability to induce polymorphonuclear leukocyte (PMN) chemiluminescence (CL) and superoxide (O2-) generation and their susceptibility to phagocytosis by PMNs. P+ strains opsonized in normal human serum (NHS) induced significantly greater CL than did P- strains (500 X 10(5) +/- 112 X 10(5) versus 242 X 10(5) +/- 65 X 10(5) total counts per 60 min; P less than 0.001) when reacted with normal PMNs. Contributions of immunoglobulin and complement to CL activity in these mixtures were shown by findings of lower overall levels of CL when hypogammaglobulinemic serum or heat-inactivated NHS was used to opsonize either P+ or P- organisms. Results obtained with mixtures of hypogammaglobulinemic plus adsorbed heat-inactivated NHS (with P+ or P- organisms) suggested a role for an antipilus antibody in the enhancement of CL by these strains. NHS-opsonized P+ strains also induced significantly greater (P less than 0.002) O2- generation than did P- strains (2.83 +/- 0.08 versus 1.94 +/- 0.14 nmol of ferricytochrome c reduced per 10 min/10(6) PMN). Comparable ingestion of P+ or P- strains opsonized in NHS by PMNs was demonstrated by a radiolabeled uptake technique and transmission electron microscopy, and primary granule release (beta-glucuronidase) was comparable during ingestion of P+ or P- strains. The basis for the observed enhanced capacity of P+ Hib to stimulate PMN oxidative metabolism as compared with P- organisms is uncertain. Possible clinical implications of these findings deserve further study.     

Regulation of human neutrophil type 3 complement receptor (iC3b receptor) expression during phagocytosis of Staphylococcus aureus and Escherichia coli.

Human neutrophils (PMN) express a receptor for iC3b, a cleavage product of C3b. CR3 is an important receptor for phagocytosis of opsonized bacteria and its expression is enhanced by cell activation. We examined PMN CR3 expression during phagocytosis using flow cytometry and a CR3-specific monoclonal antibody. After 30 min phagocytosis of opsonized S. aureus and E. coli, CR3 expression increased to 151% and 221% of controls, respectively. Unopsonized S. aureus had no effect on CR3; however, unopsonized E. coli enhanced CR3 expression despite not being phagocytosed. Time-kinetic studies indicated a rapid initial fall in CR3 after addition of bacteria to PMN, followed by enhanced expression within 5-10 min. The initial fall in CR3 probably represented CR3 internalization rather than receptor destruction, as superoxide dismutase, catalase and protease inhibitors had no effect on this. Correlation of CR3 expression with the PMN oxidative response, measured with the intracellular fluorescent probe DCF-DA, demonstrated a dichotomy. Opsonized S. aureus and E. coli caused an oxidative response from PMN but unopsonized E. coli, which caused significant CR3 up-regulation, did not. CR3 up-regulation with unopsonized and opsonized E. coli was markedly inhibited by Polymyxin B, suggesting a role for endotoxin. These experiments indicate that CR3 expression can be regulated during phagocytosis, and the mechanisms responsible are distinct from those involved in the oxidative burst. CR3 up-regulation following exposure to bacteria in vivo may enhance neutrophil function at sites of infection.     

Regulation of neutrophil functions by elastase-generated IgG fragments.

IgG is split by neutrophil elastase into Fc and Fab fragments. These IgG fragments influence the functions of stimulated neutrophils such as chemotaxis, oxidative burst, and enzyme release. FMLP stimulated leukocyte chemotaxis is specifically inhibited by the elastase generated Fc fragments. Seven nmol Fc/10(6) PMN totally inhibit the chemotaxis stimulated by 16 to 125 nM FMLP. Native IgG and Fab fragments show no effect. FMLP-stimulated superoxide anion generation is specifically inhibited by Fc fragments with half maximal inhibition by 1.2 nmol/10(6) PMN. The generation of hydrogen peroxide is concomitantly stimulated, resulting in a superoxide dismutase-like effect. FMLP-stimulated elastase and myeloperoxidase release are enhanced by Fab fragments (10 nmol/10(6) PMN) to 206 and 155%, respectively, of reference values by 25 nM FMLP, while Fc and native IgG stimulate to a less extent. Consequently, elastase-generated Fc fragments have an inhibitory effect on inflammation by reducing chemotaxis and oxidative burst of stimulated neutrophils. The release stimulating activity of Fab fragments results in an up-regulation of elastase induced IgG degradation.     

Generation of hydrogen peroxide by Candida albicans and influence on murine polymorphonuclear leukocyte activity.

Iodide fixation by murine polymorphonuclear leukocytes (PMN) incubated with viable Candida albicans blastoconidia increases directly with yeast cell concentration up to about 3 x 10(6) cells per ml, but above this concentration bound activity declines dramatically. To understand the basis for this decline, we examined the oxidative metabolism of fungi and stimulated PMN and found some remarkable similarities between these cell types. Both produced 14CO2 when incubated with [1-14C]glucose, both reduced cytochrome c, and both fixed radiolabeled iodide, although the fungi required exogenous lactoperoxidase. In dose-response experiments, iodination by fungi with lactoperoxidase was identical to that with PMN, i.e., the maximum bound activity occurred in cultures with 10(6) to 3 x 10(6) blastoconidia per ml. Iodination by fungi with lactoperoxidase was reduced when blastoconidia were incubated at 25 degrees C or in the presence of catalase and the metabolic inhibitors rotenone, antimycin A, and 2-deoxyglucose. Results from assays for oxidation of scopoletin and o-dianisidine showed that 10(6) blastoconidia in 1.0 ml of medium released 0.5 to 0.7 nmol of H2O2 after 1 h, but 3 X 10(6) and 10(7) cells released significantly less H2O2. These results suggest that iodide fixation by PMN and low numbers of fungal cells may reflect a cooperative effort, with fungi generating some H2O2 that reacts with the myeloperoxidase released from the PMN. With high concentrations of blastoconidia, H2O2 activity appeared to be specifically inhibited, possibly to protect fungal cells from damage.     

Alteration of polymorphonuclear leukocyte activity by viable Candida albicans.

The response of human polymorphonuclear leukocytes (PMN) to blastospores and pseudo-hyphae of the opportunistic fungus Candida albicans has been studied in vitro and in vivo. Of the fungicidal mechanisms elucidated thus far, the myeloperoxidase-hydrogen peroxide-halide system appears to be most effective against cells of this fungus. In our studies on the interaction between murine PMN and blastospores, we assayed the release of H2O2 by PMN incubated with viable or killed, unopsonized or opsonized blastospores by using two assay systems, lysis of murine erythrocytes and oxidation of scopoletin. Our results showed that PMN released increasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized killed blastospores, but released decreasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized viable blastospores. The oxidative metabolic burst by PMN in the presence of viable or killed blastospores was also measured by using reduction of nitroblue tetrazolium and chemiluminescence. Viable blastospores stimulated a stronger metabolic burst than killed blastospores, suggesting that PMN respond to live blastospores more vigorously than killed blastospores; however, live blastospores appear to alter or inhibit the release of H2O2 by PMN.     

Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes

Uropathogenic strains of Escherichia coli bearing mannose-sensitive (type 1) fimbriae promote a unique pattern of degranulation from human polymorphonuclear leukocytes (PMN). Significant quantities of the primary (1 degree) and tertiary (3 degree) granule markers, neutral protease-myeloperoxidase and N-acetyl-beta-D-glucosaminidase, respectively, were released by PMN in a dose- and time-dependent manner when stimulated by these defined bacterial strains. Organisms bearing mannose-resistant (P) fimbriae promoted release of only the secondary (2 degree) granule marker, vitamin B12-binding protein. When this pattern of degranulation was compared to that produced by PMN in response to a variety of soluble and particulate stimuli, only the calcium ionophore A23187 similarly triggered 1 degree and 3 degree granule marker release. All the other stimuli tested--zymosan, serum-treated and unopsonized; n-formylmethionyl-leucyl-phenylalanine; and phorbol myristate acetate--promoted release of only the 2 degree granule marker. These results demonstrate selectivity of PMN degranulation in response to a number of transmembrane signals. In addition, the capacity of E. coli to promote PMN degranulation is dependent on its phenotypic fimbrial expression, a surface characteristic which correlates significantly with its relative surface hydrophobicity as measured by binding to octyl Sepharose. Those bacteria demonstrating the greatest hydrophobicity were capable of triggering discharge of all three granule marker proteins. Thus, the mannose-sensitive fimbriae of uropathogenic E. coli may contribute significantly to their potential pathophysiologic role in renal scarring.     

Mannose-sensitive interaction of Escherichia coli with human peripheral leukocytes in vitro.

The ability of Escherichia coli which possess or lack mannose-sensitive adherence factors (adhesins) to associate with human peripheral leukocytes in vitro in the absence of serum was studied. E. coli 19+, which have mannose-sensitive adhesins, were derived from E. coli strain 19 by culturing in static Trypticase soy broth at 37 degrees C. E. coli 19-, which lack mannose-sensitive adhesins, were derived from E. coli 19 by culturing in agitated Trypticase soy broth at 30 degrees C. E. coli 19+ attached to leukocytes and stimulated the release of lysozyme but not beta-glucuronidase or lactate dehydrogenase. In contrast, E. coli 19- showed poor attachment to the leukocytes and failed to stimulate lysosomal enzyme release. During a 60-min incubation with the leukocytes, the number of viable 19+ organisms decreased, whereas the number of viable 19- remained constant. Purified type 1 pili from E. coli 19+ agglutinated the leukocytes but did not stimulate lysosomal enzyme release. Pretreatment of leukocytes with type 1 pili failed to prevent the adherence of E. coli 19+. The association of 19+ with leukocytes and subsequent release of lysozyme could be blocked by alpha-methyl-D-mannoside but not by equivalent concentrations of dextrose and sucrose. These results show that mannose-sensitive adhesins on E. coli mediate association of the organisms with leukocytes in the absence of serum components. The identity of the adhesins involved in leukocyte association has yet to be determined.     

Mannose-sensitive stimulation of human leukocyte chemiluminescence by Escherichia coli.

Escherichia coli organisms with mannose-sensitive adherence factors (adhesins) are known to associate with human peripheral leukocytes (WBCs) in vitro in the absence of serum. To determine whether the WBC respiratory burst is activated during the interaction with E. coli, WBC chemiluminescence was measured. E. coli with mannose-sensitive adhesins stimulated a sharp burst of chemiluminescence which peaked 15 to 30 min after the bacteria and WBCs were mixed. Stimulation of chemiluminescence could be abrogated by including 10 mM alpha-methyl-D-mannoside in the test suspension. The addition of alpha-methyl-D-mannoside up to 20 min after the E. coli and WBCs were combined caused a rapid decrease in chemiluminescence. E. coli stimulation of chemiluminescence could not be inhibited by pretreating the WBCs with purified type 1 pili (fimbriae). E. coli lacking mannose-sensitive adhesins failed to stimulate chemiluminescence. The results emphasize the importance of mannose-sensitive adhesins in the association of E. coli with WBCs and suggest that the E. coli-WBC interaction system may be a useful tool for studying the mechanisms involved in the activation of the respiratory burst during phagocytosis.     

Cytosolic calcium changes in individual neutrophils stimulated by opsonized and unopsonized Candida albicans hyphae.

Previous experiments suggest the critical central role of the neutrophil (PMN) respiratory burst in the prevention and containment of disseminated candidiasis. A rise in cytosolic free calcium concentrations ([Ca2+]i) has been documented as an early event after PMN stimulation which is involved in the subsequent genesis of microbicidal and inflammatory respiratory burst products. [Ca2+]i were therefore determined in individual PMN, loaded with the fluorescent calcium probe fura-2 as they attached to and spread over serum-opsonized or unopsonized Candida albicans hyphae, particles that are too big to be completely ingested. After contact between hyphae and PMN, the PMN rapidly spread over hyphal surfaces. Although both opsonized and unopsonized hyphae stimulated similar magnitudes of peak median increases in PMN [Ca2+]i, the kinetics of responses differed; median [Ca2+]i peaked within 1 min after contact with opsonized hyphae versus 4 min after contact with unopsonized hyphae. Moreover, a detectable calcium transient did not invariably follow contact and spreading of each individual PMN over a hyphal surface. In contrast to patterns seen after stimulation of PMN with opsonized zymosan, in which [Ca2+]i is greatest in the periphagosomal region, there was a more uniform distribution throughout the cytoplasm in PMN stimulated with the noningestable hyphae. These alterations in the early patterns and timing of PMN stimulation may reflect analogous differences in subsequent events which control the efficiency and specificity of microbicidal responses to uningestible hyphae and which also determine whether host tissues are damaged by the generation of toxic PMN activation products.     

Pneumocystis carinii-induced activation of the respiratory burst in human monocytes and macrophages.

Human monocytes and monocyte-derived macrophages were studied for their ability to phagocytose Pneumocystis carinii and produce superoxide (O2-) during the process. One x 10(6) freshly isolated monocytes, incubated with 0.1-3.75 x 10(6) P. carinii cysts, increased O2- production in a dose-related way. Antibodies were essential for the process since opsonized, but not unopsonized, pneumocysts induced O2- production significantly above the response obtained by lung tissue from rats (10.7 and 4.9 versus 3.0 fmol/cell per 90 min). The difference between pneumocysts opsonized in untreated versus complement-depleted serum was not significant (10.7 versus 12.6 fmol/cell per 90 min). Monocyte-derived macrophages also activated the respiratory burst when stimulated with pneumocysts, and this effect could be significantly increased, from 4.2 to 8.8 fmol/cell per 90 min, when cells were primed with interferon-gamma (IFN-gamma). Cells primed with IL-3 also increased O2- production, though to a lesser extent. In contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF) had only a small effect on the respiratory burst in cells stimulated with P. carinii. Priming with IFN-gamma increased the rate of phagocytosis in macrophages. After incubation for 90 min or more, however, the percentage of cells with phagocytic vacuoles was only slightly higher in IFN-gamma-primed cells. When examined by electron microscopy (EM), most vacuoles contained partially or totally degraded pneumocysts. In conclusion, we have demonstrated the ability of monocytes and monocyte-derived macrophages to ingest and degrade pneumocysts, activating the respiratory burst during the process.     

Susceptibility of porcine intestine to pilus-mediated adhesion by some isolates of piliated enterotoxigenic Escherichia coli increases with age.

Two porcine isolates of enterotoxigenic Escherichia coli (ETEC) (serogroup O157 and O141) derived from fatal cases of postweaning diarrhea and lacking K88, K99, F41, and 987P pili (4P- ETEC) were tested for adhesiveness to small-intestinal epithelia of pigs of different ages. Neither strain adhered to isolated intestinal brush borders of newborn (1-day-old) pigs in the presence of mannose. However, mannose-resistant adhesion occurred when brush borders from 10-day- and 3- and 6-week-old pigs were used. Electron microscopy revealed that both strains produced fine (3.5-nm) and type 1 pili at 37 degrees C but only type 1 pili at 18 degrees C. Mannose-resistant in vitro adhesion to brush borders of older pigs correlated with the presence of fine pili. These strains produced predominantly fine pili in ligated intestinal loops of both older and newborn pigs, but adherence was greater in loops in older pigs. Immunoelectron microscopic studies, using antiserum raised against piliated bacteria and absorbed with nonpiliated bacteria, of samples from brush border adherence studies revealed labelled appendages between adherent bacteria and intestinal microvilli. Orogastric inoculation of pigs weaned at 10 and 21 days of age indicated significantly (P less than 0.001) higher levels of adhesion by the ETEC to the ileal epithelia of older pigs than to that of younger ones. We suggest that small-intestinal adhesion and colonization by these ETEC isolates is dependent on receptors that develop progressively with age during the first 3 weeks after birth. Furthermore, our data are consistent with the hypothesis that the fine pili described mediate intestinal adhesion by the 4P- ETEC strains studied.     

Differential effects of lithocholate on rat neutrophil activation

Neutrophils (PMNs) may be exposed to high concentrations of biliary products during cholestasis and other hepatic disorders. We have previously reported that bile and certain bile salts enhance superoxide (O2-) release from neutrophils activated with phorbol myristate acetate (PMA) (Dahm et al.: Toxicol. Appl. Pharmacol. 95, 82, 1988), suggesting that PMN oxidative metabolism might be altered in toxicoses or disease states characterized by elevations in serum bile salts and other biliary products. In the present study, we characterized the priming effect of lithocholate for O2- release and also examined the effects of lithocholate on enzyme release from PMNs. PMNs preincubated with lithocholate at concentrations which did not directly stimulate O2- release (3-100 microM) and activated with PMA released greater amounts of O2- than controls exposed to PMA alone, illustrating a priming effect. O2- release from lithocholate-primed PMNs rose sharply between 5 and 10 min after PMA addition and then ceased between 10 and 30 min. The priming effect of lithocholate toward PMA-activated PMNs was reduced approximately 50% by washing PMNs after lithocholate addition and was not dependent on extracellular Ca2+, although removal of Ca2+ from the incubation buffer enhanced the cytotoxicity of lithocholate toward PMNs. In Ca2(+)-supplemented medium, lithocholate primed PMNs for O2- release when formyl-methionyl-leucyl-phenylalanine (FMLP, 10(-8)-10(-6) M) or calcium ionophore, A23187 (10(-7) or 10(-6)M), was used to activate PMNs. Lithocholate (100 microM) by itself had only marginal effects on release of lysozyme or beta-glucuronidase from PMNs. However, lithocholate (100 microM) inhibited beta-glucuronidase release from FMLP-stimulated PMNs to near-baseline levels. When FMLP was added to PMNs prior to lithocholate, beta-glucuronidase release was not reduced as it was when the order of addition was the reverse. Lithocholate had no effect on PMA-stimulated lysozyme release. These results indicate that lithocholate has different actions on PMN O2- release and enzyme release and suggest that lithocholate might exert its action on the PMN plasma membrane.     

Oxidative metabolism of neonatal and adult rabbit lung macrophages stimulated with opsonized group B streptococci.

We compared the oxidative metabolism of alveolar macrophages (AM) from adult and neonatal (1- and 7-day-old) rabbits before and after their in vitro exposure to type Ia group B streptococci (GBS) opsonized with immune rabbit serum. Nonstimulated AM from 1-day-old, 7-day-old, or adult rabbits consumed O2 at a rate of 17 to 20 nmol/10(6) AM per 10 min under basal conditions and released minimal amounts of superoxide (O2-) into the medium. Approximately 80% of this basal respiration was of mitochondrial origin, based on its inhibition by NaCN. Exposure to GBS opsonized with immune rabbit serum stimulated O2 consumption approximately half as effectively in the neonatal AM as in the adult AM. Little O2- was released into the medium unless the cells were pretreated with dihydrocytochalasin B. Under such conditions, 1-day-old, 7-day-old, and adult AM released 3.6, 5.3, and 13.9 nmol of O2-/10(6) AM per 10 min, respectively. The uptake of opsonized GBS by 1-day-old AM was not affected by 1 mM NaCN, whereas phagocytosis by adult AM was substantially reduced under these conditions. Overall, our findings suggest that neonatal AM have less-well-developed postphagocytic oxidative metabolic responses and release less superoxide after exposure to opsonized GBS than do adult AM. They also demonstrate that the energy for phagocytosis is derived principally from mitochondrial metabolism in adult AM but not in neonatal AM. We conclude that metabolic differences between neonatal and adult AM may contribute to neonatal pulmonary susceptibility to GBS infections and account, in part, for the ability of GBS to succeed as neonatal pulmonary pathogens.     

Effect of sub-minimal inhibitory concentrations of ciprofloxacin and fleroxacin on the bacterial capsular antigen and opsonophagocytosis by human polymorphonuclear leukocytes

We have studied the effect of sub-minimal inhibitory concentrations (sub-MIC) of ciprofloxacin and fleroxacin on capsulated (K+) and non-capsulated (K-) Gram-negative bacilli (Escherichia coli O1:K1, O7:K1, O1:K-, O7:K-, and Klebsiella oxytoca) as well as on Staphylococcus aureus and we investigated the interaction of antibiotic pretreated bacteria with human serum and polymorphonuclear leukocytes (PMN). Following overnight growth in the presence of 1/2 MIC of the antibiotics, bacteria were opsonized in human serum and incubated with PMN. Opsonophagocytosis was quantified as the ratio of uptake by PMN of radioactively labeled bacteria. Ciprofloxacin and fleroxacin enhanced the phagocytosis rate of E. coli K+ strains (control 5-10%; 1/2 MIC of ciprofloxacin and fleroxacin 70-80%) of K. oxytoca (control O-2%; 1/2 MIC of ciprofloxacin and fleroxacin 35-40%) as well as of Staph. aureus (control 5-10%; 1/2 MIC of the antibiotics 35-40%). Opsonophagocytosis of K- strains was not altered. The enhancement of opsonophagocytosis was a complement dependent process. Exposure of capsulated E. coli to ciprofloxacin as well as to fleroxacin resulted in decreased amounts of the capsular antigen.     

Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide, by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS-LBP, and LPS-HDL complexes to activate a number of cellular responses have been compared. LPS-LBP and LPS-HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS-LBP and LPS-HDL complexes differed in their ability to prime PMN O2- production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2- production is minimal (less than 1 nmol O2-/10(6) PMN/10 min) after exposure to 10(-7) M FNLPNTL can be primed with 0.1-100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2-/10(6) PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMS to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.     

Opsonization of Staphylococcus aureus protects endothelial cells from damage by phagocytosing polymorphonuclear leukocytes.

When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.     

Stimulus-dependent inhibition of superoxide generation by prostaglandins

Infiltrating phagocytes generate superoxide anion (O2-) and prostaglandin (PG) at sites of inflammation. Thus PG-O2- interactions may be important to the initiation and control of inflammation. PGE1, PGE2, and PGD2 inhibit O2- generation (as measured by superoxide dismutase-inhibitable reduction of ferricytochrome c) in a dose-dependent manner (10(-6)-10(-9) M) when human peripheral blood polymorphonuclear leucocytes (PMN) are stimulated with 10(-7) M of the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). These PG did not alter O2- generation when PMN were stimulated with 0.1 microgram/ml phorbol myristate acetate (PMA) or 1 mg/ml serum-treated zymosan (STZ). Increments of cyclic AMP (cAMP) (peak: fourfold) in PGE1, PGE2, and PGD2 treated PMN stimulated with PMA or STZ (in which O2- was not reduced) were similar to those in PG-treated PMN stimulated with FMLP (in which O2- was reduced markedly). High concentrations of theophylline and dibutyryl cAMP reduced FMLP and STZ stimulated O2- generation but had no effect on PMA stimulation, suggesting that the stimuli induce different sensitivities to the effects of cellular cAMP. PGF2 alpha had little effect on O2- generation or cAMP levels regardless of the stimulus. PGE1 did not inhibit binding of FML(3H)P to PMN and did not scavenge O2- anions. Therefore the effect of PG on O2- production is dependent on the specific stimulator and an increased concentration of cAMP in activated PMN is by itself not sufficient to limit O2- generation induced by all stimuli.     

Characteristics of binding of Escherichia coli serotype O157:H7 strain CL-49 to purified intestinal mucin.

Purified rat intestinal mucin was used as a model mucin to study the binding of Escherichia coli serotype O157:H7, a human pathogen associated with outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. Of six O157:H7 strains, only one strain (designated CL-49) bound to rat (and other) intestinal mucins by a specific and saturable process. Binding was observed only after the bacteria were serially passaged to promote the expression of type 1 pili (fimbriae). Several other type 1-piliated E. coli strains, however, did not bind to mucin. Binding of E. coli CL-49 was inhibited by D-mannose and short oligomannosyl derivatives, particularly Man-alpha-1,3-Man, Man-alpha-1,2-Man, and Man-alpha-1,3-Man-beta-1,4-N-acetylglucosamine. Other inhibitors of binding included p-nitrophenol (10(-4) M), heating at 60 degrees C (to remove pili), an antibody to type 1 pili, and purified type 1 pili of E. coli CL-49 used as hapten inhibitors. A comparison of the hydrophobicity of piliated E. coli CL-49 with other type 1-piliated E. coli strains indicated that the former strain was much more hydrophobic than the others. These findings indicate that highly purified intestinal mucins possess specific mannosyl receptor sites for bacterial type 1 pili on E. coli CL-49, but that strong hydrophobic interactions between the mucin and the pili stabilize the mannose-dependent binding process. We speculate that the mucin receptors for type 1 pili reside in oligosaccharides of the 118-kilodalton "link" glycopeptide, since this is the only mucin component known to contain mannose.     

Inhibition of Neutrophil Chemotaxis by Elastase-Generated IgG Fragments

IgG1 is cleaved in vitro by granulocyte elastase into Fc and Fab fragments. The elastase-specific Fc fragment has been previously detected in vivo. Biological activity of the fragments has been described in modulating neutrophil oxidative metabolism and enzyme release. To investigate further effects granulocyte chemotaxis (CT) was tested. The CT was assayed in Boyden chambers and the chemotactic index (CI) was calculated which represents the mean distance travelled by the activated cells. Stimulation of leucocyte CT by casein, activated serum and FMLP gives maximal values of delta CI = 46.7, 26.4 and 7.2 microns, respectively. Native IgG1 and the elastase-produced IgG fragments do not stimulate leucocyte CT. FMLP-stimulated CT is specifically inhibited by the elastase-produced Fc fragments. Addition of 7 nmol Fc to stimulus concentrations of 16 to 125 nM FMLP results in total inhibition of chemotaxis demonstrated by CI values which are lower than those for unstimulated cells. The inhibition of CT is concentration dependent in the range of 2 to 7 nmol Fc/10(6) PMN. Number and affinity of FMLP receptors are not influenced by Fc fragments, so Fc binds neither to FMLP nor the FMLP receptor. CT stimulated by casein shows a large portion of chemokinesis. Only at suboptimal casein concentrations do Fc and IgG have an inhibitory effect on CT (0.63 mg casein/ml, 10 nmol peptide/10(6) PMN). C5a-stimulated CT is not influenced by IgG or IgG fragments which indicates that the samples are not cytotoxic. So the FMLP and casein-stimulated CT is specifically inhibited by the elastase-produced Fc fragments in a low concentration range.     

Suppression of human lymphocyte proliferation by activated neutrophils or H2O2: surviving cells have an altered T helper/T suppressor ratio and an increased resistance to secondary oxidant exposure

Either activated neutrophils (PMNs) or nanomole amounts of reagent hydrogen peroxide (H2O2) were found to cause catalase-reversible suppression of concanavalin A (Con A)-stimulated human lymphocyte proliferation. Suppression required PMN activation and occurred with PMN/lymphocyte ratios found in peripheral blood. Inhibition by reagent H2O2 occurred with 10-40 nmol H2O2/1 X 10(6) lymphocytes, a noncytolethal amount which is produced readily by PMA-activated PMNs. Lymphoblasts recovered from suppressed cultures were two- to fourfold less sensitive than control lymphoblasts to a second exposure to H2O2. These relatively H2O2-resistant lymphoblasts also scavenge H2O2 at higher rates than do control lymphoblasts. Progeny lymphocytes from suppressed cultures contain an unusually high percentage of T helper cells with a concomitant decrease in T suppressors. These studies demonstrate a potential immunoregulatory role for phagocyte-released oxidants, especially H2O2, and suggest a possible additional mechanism to explain the excess of T helpers observed in situations of chronic inflammation.