Normal Human Polymorphonuclear Leukocytes Research Articles

Omeprazole Induces Apoptosis in Normal Human Polymorphonuclear Leucocytes

We investigated in vitro apoptosis in human polymorphonuclear neutrophils (PMN) induced by omeprazole. This drug, both in the native (OM) and acidified (OM-HCl) form, is a potent inducer of PMN apoptosis. The effect is time- and dose-dependent. OM-HCl is more efficient than OM in inducing PMN apoptosis. In fact, after 24 h incubation in vitro at 1 x 10(-4) M OM-HCl induces apoptosis in 70% of the cell population compared to 37% induced by OM. Apoptosis induced by both forms of the drug is caspase dependent being significantly reduced by pretreating cells with the caspase 3 inhibitor (DEVDH-CHO). However, some differences in the apoptosis mechanisms between the two forms of the drug seem to exist because PMN treatment with the specific caspase 8 inhibitor (Z-IETD-FMK) only blocks OM-HCl mediated apoptosis. We observed cleavage of caspase 8 only in the cells incubated with OM-HCl while the executioner caspase 3 was activated with both forms of the drug. Furthermore, pretreatment with GM-CSF, a known activator of intracellular survival pathways in PMN, partially protected cells from OM-HCl induced apoptosis but did not contrast the apoptotic effect of OM. Cysteine cathepsin proteases also seem involved in the apoptotic mechanism of both drug forms since the specific inhibitor E64d gave a significant protection. To verify if OM-HCl induced apoptosis was dependent on the sulfenamide bound with the cell sulfhydryl groups we used molecules with thiol groups such as beta-mercaptoethanol (beta-ME) and reduced glutathione (GSH). Reactions of OM-HCl with cellular sulfhydryl groups are strongly involved in both the triggering and evolving phase of the apoptotic mechanism since significant protection from apoptosis was obtained when PMN were pretreated for 1 h with beta-ME (lipid-permeable) or GSH (lipid-impermeable). These results show that OM and OM-HCl induce apoptosis in human PMN and suggest that the second binds the sulfhydryl groups, present on the cell membrane, to then penetrate the cell thus causing a further significant increase in apoptosis. OM-induced PMN apoptosis during the treatment of gastric inflammatory disease could be an advantage for the resolution of the phlogosis state. However, this aspect should be further elucidated to assess the optimal therapeutical regimen for gastric diseases which are related to infective agents.

Antibody to capsular polysaccharide of Streptococcus pneumoniae at the time of hospital admission for Pneumococcal pneumonia.

IgG to capsular polysaccharide (CPS) of Streptococcus pneumoniae is thought to provide the greatest degree of protection against pneumococcal disease. Serum obtained at hospital admission from 14 (27%) of 51 patients with bacteremic pneumococcal pneumonia and 11 (37%) of 30 with nonbacteremic pneumococcal pneumonia contained IgG to CPS of the infecting serotype; these percentages are similar to the prevalence of IgG to CPS in a control population. However, when compared with antibody from healthy adults, this IgG had far less capacity to opsonize the infecting pneumococcal serotype for phagocytosis in vitro by normal human polymorphonuclear leukocytes or to protect mice against experimental challenge. Failure to opsonize correlated closely with failure to protect mice, and each of these parameters correlated well with poor avidity for CPS. Future vaccine studies may need to examine the functional capacity of antibodies as a surrogate for infection, in addition to measuring their concentration in serum.

Influence of IgG1 and IgG2 antibodies to streptococcal group A carbohydrate on neutrophil chemiluminescence

IgG antibodies to streptococcal group A carbohydrate (A-CHO) were isolated from normal human serum and from pooled Cohn fraction II using N-acetyl-D-glucosamine affinity chromatography columns. They consisted of the subclasses IgG1 and IgG2 in variable proportions depending on the pH of the elution buffer and on the subclass composition of anti-A-CHO in the starting material. Streptococcal group A particles exposing the antigen A-CHO were opsonized with various concentrations of these antibodies and incubated with normal human polymorphonuclear leukocytes (PMN). The luminol-enhanced chemiluminescence (CL) elicited with these particles was recorded over a 60-min period. With 12 of the 14 antibody preparations tested, the observed CL signal was dose-dependent. When used at comparable concentrations for opsonization, the antibodies enriched in IgG1 induced markedly stronger CL signals than those consisting predominantly of IgG2. It was concluded that the observed CL differences reflect mainly the preferential recognition of IgG1 molecules by Fc gamma receptors of PMN.

Polymorphonuclear Leukocyte-Induced Detachment of Cultured Epidermal Carcinoma Cells from the Substratum

In an in vitro study, intended to develop tumoricidal therapy with the use of human leukocyte, an interesting phenomenon was found. Normal human polymorphonuclear leukocytes (PMN) plated on a cell line established from malignant trichilemmal cyst (DJM-1), a kind of squamous cell carcinoma, in a serum-free media and incubated for 48 h induced the detachment of DJM-1. The detachment was more extensive as the number of PMN increased. The detachment rate was 97.0% when the number of PMN and DJM-1 in a well was in a ratio 2.4:1 and the viability of detached DJM-1 was 96.5%. Two kinds of proteinase inhibitors, especially the inhibitor of neutrophil elastase, fetal bovine serum, and monoclonal anti-laminin antibody inhibited the detachment significantly. Furthermore, when PMN were seeded in a chamber with a filter membrane bottom to prevent direct contact with DJM-1, DJM-1 detachment decreased to 14.2%. In view of these results, the following mechanism was postulated. Activated by their adhesion to DJM-1, especially between laminin receptor on PMN and laminin on DJM-1, PMN secreted proteinases, resulting in DJM-1 detachment. This phenomenon might be an expression of cytotoxicity of PMN to cancer cells, because cultured cancer cells of epithelium origin such as DJM-1 can grow only after they are firmly attached to the substratum. This phenomenon, in turn, may explain the final step in the induction of epidermal-dermal separation in subepidermal bullous diseases with PMN infiltration such as bullous pemphigoid and dermatitis herpetiformis if we could regard DJM-1 as normal keratinocyte.

Lysosomal enzyme release from normal human polymorphonuclear leukocytes in response to particulate stimuli

The microbicidal capacity of polymorphonuclear (PMN) leukocytes of normal human subjects was evaluated by estimating the release of lysosomal enzymes from normal cells and cells rendered incapable of ingesting particles by treatment with cytochalasin B, on exposure to a particulate stimulus-serum treated zymosan (STZ). The release of lysosomal enzymes namely beta-glucuronidase, lysozyme, acid phosphatase, alkaline phosphatase was found to be 16.28%, 27.84%, 20.54%, 15.40% respectively. The cells rendered incapable of ingesting particles showed enhanced response. The percentage release of beta-glucuronidase, lysozyme, acid and alkaline phosphatase was increased to 20.58%, 37.95%, 25.87% and 33.04% respectively. This type of response in cytochalasin B treated PMN leukocytes is due to the conversion of these cells into “secretory cells”, which release lysosomal enzymes outside the cell, by the process of reverse endocytosis.

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer.

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. avium-intracellulare). Other possible applications of this method are discussed.

Degradation of gonococcal peptidoglycan by granule extract from human neutrophils: demonstration of N-acetylglucosaminidase activity that utilizes peptidoglycan substrates.

The degradation of purified Neisseria gonorrhoeae peptidoglycan (PG) by granule extract derived from normal human polymorphonuclear leukocytes was examined. Hen egg lysozyme-resistant, extensively O-acetylated [3H]PG (O-PG) from strain FA19 and lysozyme-sensitive, non-O-acetylated [14C]PG (non-O-PG) from strain RD5 (each containing label in both glucosamine and muramic acid) were mixed and incubated with granule extract at pHs 4.5, 5.5, and 6.5. The rate of degradation of O-PG was uniformly slower than that of non-O-PG in the same tube, but ultimately, even the O-PG was rendered completely soluble. Molecular-sieve high-performance liquid chromatography revealed that both PGs were degraded by granule extract at the pH values tested to disaccharide peptide monomers and peptide-cross-linked oligomers, reflecting the action of human lysozyme. Of particular interest was the appearance of a peak containing free N-acetylglucosamine which was quite prominent in reaction mixtures at pH 4.5, less prominent at pH 5.5, and not detectable at pH 6.5. Free N-acetylglucosamine was not released from control PG samples at any pH in the absence of granule extract. Treatment of purified gonococcal PG monomers with granule extract at pH 4.5 yielded exclusively free N-acetylglucosamine and muramyl peptides with no N-acetylglucosamine. These data suggest that granule extract contains a previously undescribed pH-dependent N-acetylglucosaminidase with specificity for PG as well as an N-acetylmuramidase activity that degrades O-PG less efficiently than it does non-O-PG.

Impaired polymorphonuclear leukocyte function in biliary atresia:Role of bilirubin and bile acids

Our previous study suggested that impaired bactericidal activity of polymorphonuclear leukocytes in patients with biliary atresia might be due to decrease in superoxide-generating activity and that serum factors may play an important role. In the present study, analysis of the patients' serum revealed that, among 15 bile acids, glycine or taurine conjugated chenodeoxycholic acid and cholic acid were markedly elevated. To examine the effects of bilirubin and these bile acids and conjugated lithocholic acids on the leukocyte function, we measured cytolysis, phorbol myristate acetate-induced superoxide generation, and myeloperoxidase activity, using normal human polymorphonuclear leukocytes. Bilirubin ranging from 5 to 20 mumol/L was cytolytic and more potently inhibited the superoxide generation. The inhibition by bilirubin was also observed in the presence of 10% human serum or 2.0% human serum albumin, though the cell viability was almost completely preserved. On the other hand, conjugated chenodeoxycholic acids and cholic acids, ranging from 0.5 to 1.5 mmol/L or conjugated lithocholic acids, ranging from 0.02 to 0.05 mmol/L, did not inhibit the superoxide generating activity, though the conjugated chenodeoxycholic acids and lithocholic acids did induce cytolysis. Myeloperoxidase activity was little affected, except in the case of conjugated lithocholic acids. These results suggest that in patients with biliary atresia, bilirubin, probably the unconjugated form, more than bile acids might be one of the influential factors in the suppression of bactericidal activity of polymorphonuclear leukocytes, by inhibiting the superoxide-generating activity.

Tyrosine kinase and phosphotyrosine phosphatase activity in human promyelocytic leukemia cells and human polymorphonuclear leukocytes

Although an increase in protein phosphorylation on tyrosine was first noted as a result of cell transformation or the application of growth factors to cells, recent reports have shown high levels of tyrosine kinases in nondividing tissues. For that reason, we have investigated whether normal human polymorphonuclear leukocytes (PMN) contain tyrosine kinase and phosphatase activity. Using a copolymer of glutamine: tyrosine as a substrate for the phosphotransferase reaction, we have demonstrated that PMN contain a cytosolic tyrosine kinase activity that elutes as a single peak from Sephacryl S-200 chromatography and has a molecular weight of 70 kilodaltons. Human promyelocytic leukemia cells (HL-60), contain a similar activity (as demonstrated by column chromatography), with only 25% of the activity found in PMN. This cytosolic tyrosine kinase can phosphorylate angiotensin II and a fragment of the src protein containing tyrosine 416, which suggests a similar substrate specificity to other tyrosine- phosphorylating protein kinases. In addition, we have demonstrated that PMN have double the amount of phosphotyrosine phosphatase (PTPase) activity of that found in HL-60 cells. This enzyme has a Km of 0.932 mmol/L and a Vmax of 0.355 mumol inorganic phosphate released/mg protein/min, which is similar to other cellular PTPase. Activation of PMN with f-Met-Leu-Phe and phorbol esters causes a slight but statistically significant drop in PMN PTPase activity. These results suggest that terminally differentiated myeloid cells have high tyrosine kinase and phosphatase activity, which may play a role in stimulus response coupling in the mature PMN.

Tyrosine Kinase and Phosphotyrosine Phosphatase Activity in Human Promyelocytic Leukemia Cells and Human Polymorphonuclear Leukocytes

Although an increase in protein phosphorylation on tyrosine was first noted as a result of cell transformation or the application of growth factors to cells, recent reports have shown high levels of tyrosine kinases in nondividing tissues. For that reason, we have investigated whether normal human polymorphonuclear leukocytes (PMN) contain tyrosine kinase and phosphatase activity. Using a copolymer of glutamine: tyrosine as a substrate for the phosphotransferase reaction, we have demonstrated that PMN contain a cytosolic tyrosine kinase activity that elutes as a single peak from Sephacryl S-200 chromatography and has a molecular weight of 70 kilodaltons. Human promyelocytic leukemia cells (HL-60), contain a similar activity (as demonstrated by column chromatography), with only 25% of the activity found in PMN. This cytosolic tyrosine kinase can phosphorylate angiotensin II and a fragment of the src protein containing tyrosine 416, which suggests a similar substrate specificity to other tyrosine-phosphorylating protein kinases. In addition, we have demonstrated that PMN have double the amount of phosphotyrosine phosphatase (PTPase) activity of that found in HL-60 cells. This enzyme has a Km of 0.932 mmol/L and a Vmax of 0.355 mumol inorganic phosphate released/mg protein/min, which is similar to other cellular PTPase. Activation of PMN with f-Met-Leu-Phe and phorbol esters causes a slight but statistically significant drop in PMN PTPase activity. These results suggest that terminally differentiated myeloid cells have high tyrosine kinase and phosphatase activity, which may play a role in stimulus response coupling in the mature PMN.

Actin Polymerization and Its Relationship to Locomotion and Chemokinetic Response in Maturing Human Promyelocytic Leukemia Cells

We studied actin polymerization in the HL-60 human promyelocytic leukemia cell line during induced myeloid maturation and its relationship to the rate of locomotion (ROL). The percent G-actin (of total actin) was measured by DNAase I inhibition, F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained cells, and ROL was measured by computer-assisted analysis of the tracks of individual cells. Uninduced HL-60 cells moved slowly (2.3 +/- 1.0 microns/min) and showed no change in ROL or in the state of actin polymerization when stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP). Nonstimulated cells induced to differentiate with dimethylformamide had no change in the degree of actin polymerization but exhibited a mean (m) ROL similar to normal human polymorphonuclear leukocytes (PMN) (8.6 +/- 1.4 micron/min [HL-60 cells] v 7.8 +/- 1.8 microns/min [PMN]. When induced HL-60 cells were stimulated with fMLP, actin polymerization occurred. The F-actin content increased, as determined by FACS analysis of NBD-phallacidin-stained cells, and the percentage of G-actin decreased, as determined by a 24.5% decrease in DNAase I inhibitory activity. However, induced HL-60 cells stimulated with fMLP did not increase their mROL. These studies show that, unlike normal human PMN, chemotactic peptides can cause an intracellular biochemical change that is not associated with a chemokinetic response in induced HL-60 cells. The HL-60 cell line may be a useful model to study the development of chemotactic peptide-mediated actin polymerization during myeloid cell maturation.

Actin polymerization and its relationship to locomotion and chemokinetic response in maturing human promyelocytic leukemia cells

We studied actin polymerization in the HL-60 human promyelocytic leukemia cell line during induced myeloid maturation and its relationship to the rate of locomotion (ROL). The percent G-actin (of total actin) was measured by DNAase I inhibition, F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained cells, and ROL was measured by computer-assisted analysis of the tracks of individual cells. Uninduced HL-60 cells moved slowly (2.3 +/- 1.0 microns/min) and showed no change in ROL or in the state of actin polymerization when stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP). Nonstimulated cells induced to differentiate with dimethylformamide had no change in the degree of actin polymerization but exhibited a mean (m) ROL similar to normal human polymorphonuclear leukocytes (PMN) (8.6 +/- 1.4 micron/min [HL-60 cells] v 7.8 +/- 1.8 microns/min [PMN]. When induced HL-60 cells were stimulated with fMLP, actin polymerization occurred. The F-actin content increased, as determined by FACS analysis of NBD-phallacidin-stained cells, and the percentage of G-actin decreased, as determined by a 24.5% decrease in DNAase I inhibitory activity. However, induced HL-60 cells stimulated with fMLP did not increase their mROL. These studies show that, unlike normal human PMN, chemotactic peptides can cause an intracellular biochemical change that is not associated with a chemokinetic response in induced HL-60 cells. The HL-60 cell line may be a useful model to study the development of chemotactic peptide-mediated actin polymerization during myeloid cell maturation.

Enhanced oxidative burst without interleukin 1 production by normal human polymorphonuclear leukocytes primed with muramyl dipeptides.

Purified polymorphonuclear (PMN) cells were obtained from human blood leukocytes by centrifugation on colloidal silica gradients. PMNs could be primed for PMA-triggered oxidative burst by muramyl peptide molecules (MDP) and two of its adjuvant active nonpyrogenic derivatives. The priming effect of MDP could be demonstrated after a 1-h incubation period, whereas monocytes needed an 18-h incubation to produce an enhanced response in the NBT reduction test. Only the monocyte-enriched population was able to produce IL-1 activity after muramyl peptide stimulation. Under such conditions, PMNs neither produced nor secreted IL-1-like activity, and no IL-1 inhibitor was present in the supernatant fluids. In conclusion, muramyl peptides were able to prime PMNs for oxidative burst but not to stimulate IL-1-like factor production.

Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa

Purification of a bactericidal protein (BP) from the cytoplasmic granules of normal human polymorphonuclear leukocytes (PMN) with activity against Pseudomonas aeruginosa is described. Bactericidal activity from acid extracts of a mixed granule population was purified 175-fold by a two-step chromatographic procedure. The first step, dye-ligand affinity chromatography with Matrex-Gel Orange A, was followed by cation-exchange chromatography with Bio-Rex 70 resin, and this combination routinely gave a yield near 80%. Only one peak of bactericidal activity against P. aeruginosa type I was found after each chromatographic step. Characterization of BP showed a single band with an apparent molecular weight of 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified BP was most active against the six strains of P. aeruginosa tested and against Escherichia coli B (a deep rough mutant). Purified BP killed 5 X 10(6) CFU of P. aeruginosa type I per ml at a concentration of 60 to 80 ng/ml. Proteus mirabilis and Staphylococcus aureus were both resistant to the bactericidal activity of BP. BP was shown to be glycosylated by periodic acid staining after gel electrophoresis and to have an isoelectric point near 7.5 by chromatofocusing. The amino acid composition of BP is presented.

Effect of sodium thiomalate on immune complex-induced release of lysosomal enzymes from human polymorphonuclear leucocytes

The extracellular release of β-N-acetylglucosaminidase from normal human polymorphonuclear leucocytes initiated with bovine serum albumin/anti-bovine serum albumin immune complex (15 μg/ml) was significantly inhibited (p < 0.01) by pretreatment with increasing concentrations (10−8 M, 10−7 M, 10−6 M and 10−5 M) of sodium thiolamate in a time- and dose-related fashion. Also, β-glucuronidase and β-N-acetylglucosaminidise exhibited similar responses to the effects of bovine serum albumin/anti-bovine serum albumin or sodium thiomalate. In contrast, neither bovine serum albumin/anti-bovine serum albumin nor sodium thiomalate provoked appreciable leakage of the cytoplasmic enzyme, lactate dehydrogenase. This indicates that cell integrity remains intact under the experimental conditions described.

Effect of diclofenac sodium, tolfenamic acid and indomethacin on the production of superoxide induced by N-formyl-methionyl-leucyl-phenylalanine in normal human polymorphonuclear leukocytes.

The effect of three non-steroidal anti-inflammatory drugs, diclofenac, indomethacin and tolfenamic acid, on the production of superoxide (O2-), by normal human polymorphonuclear leukocytes (PMNL) was studied, in vitro. The cells were activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and O2- production was measured as superoxide dismutase inhibitable cytochrome c reduction. Cell viability was checked with assays of liberated LDH. Concentrations of the drugs considerably higher than those of therapeutic plasma were required to inhibit O2- production. The drug concentrations producing a 50% inhibition (IC50) of total O2- production were: diclofenac 2.7 X 10(-4) M, indomethacin 4.0 X 10(-4) M and tolfenamic acid 4.2 X 10(-4) M. At drug concentrations causing a significant suppression of O2- generation, diclofenac showed a slight and tolfenamic acid a marked inhibition of [3H]FMLP binding to its cellular receptor; indomethacin has earlier been shown to inhibit FMLP-binding slightly. No dismutating activity of the drugs could be demonstrated. It is concluded that the inhibition of O2- production is due to a combined effect on FMLP binding and on cellular O2- metabolism. Because of the high drug concentrations required to inhibit O2- production, this phenomenon is obviously of little significance for the anti-inflammatory effect obtained with therapeutic doses of the drugs studied.

Relation between serum opsonic activity for Streptococcus pneumoniae and complement function in sickle cell disease.

Opsonic activity for Streptococcus pneumoniae mediated by the alternative and classic complement pathways and concomitant binding of activated C3 to the bacteria were measured in sera from children with sickle cell disease and normal siblings of similar age. Uptake of radiolabeled serotypes 7F, 10A, 15B, and 24F by normal human polymorphonuclear leukocytes and intracellular killing were the parameters used to assess opsonization. C3 fixation was quantitated by radioimmunoassay under conditions identical to those used for opsonic measurements. Both classic and alternative pathway-mediated opsonic activities were significantly reduced in a subset of patients. These alterations were associated with reduction in C3 fixation by way of the classic pathway and normal C3 fixation by way of the alternative pathway. The data implicate auxiliary serum factors rather than an intrinsic defect in the complement system in the opsonic alterations. Retrospective data were suggestive of an increased incidence of pneumococcal bacteremia occurring in association with reduction in opsonic activity.

Comparison between the luminol-dependent chemiluminescence of polymorphonuclear leukocytes and of the myeloperoxidase-HOOH system: influence of pH, cations and protein.

Abstract— Measurements of luminol‐dependent chemiluminescence (CL) from normal human polymorphonuclear leukocytes (PMNL) were carried out and the results were compared to the CL obtained from a cell‐free myeloperoxidase(MPO)‐HOOH‐system.The chemotactic peptide formylmethionyl‐leucyl‐phenyl‐alanine (fMLP) induced a bimodal luminol‐dependent CL response in PMNL, while phorbol myristate acetate (PMA) induced a one‐peak response. Calcium was found to be required for the fMLP but not for the PMA induced response. The absence of glucose reduced the PMA but not the fMLP induced PMNL CL response. Neither calcium nor glucose had any effect on the light emitted from a cell‐free MPO‐HOOH‐system.The PMNL CL response to both fMLP and PMA was affected by the pH‐value, in that at a low pH (6.3) the response was decreased and at a high pH (8.0) the response was increased. These effects were also obtained in cell‐free MPO‐HOOH‐systems. The presence of human serum albumin (HSA) totally inhibited the CL of a cell‐free MPO‐HOOH‐system while, in PMNL CL, HSA reduced only the first phase of the fMLP induced response.The results are discussed in view of the fact that CL is increasingly used as a clinical instrument to study defects in PMNL function and the effects on PMNL function of various agents such as drugs.

Resistance of Vibrio vulnificus to serum bactericidal and opsonizing factors: relation to virulence in suckling mice and humans.

Vibrio vulnificus causes soft-tissue infections, gastrointestinal disease, and severe sepsis in humans. Bacterial and host factors in virulence have remained poorly defined. We found that blood culture isolates of V. vulnificus were completely resistant to the bactericidal effects of 10% normal human serum, in contrast to soft-tissue and environmental isolates that showed a mean 2.6 log10 decline during 120 min of incubation. Following opsonization by 10% normal human serum, mean uptake of blood isolates by normal human polymorphonuclear leukocytes during 20 min of incubation in vitro was 45.8% compared with 83.2% for isolates from other sites. Blood isolates were lethal for suckling mice (mean LD50, 1.3 X 10(6)) in contrast with isolates from other sites, which were less virulent (mean LD50, 1 X 10(9)); lethality correlated well with bacteremia at 6 hr. These studies show a close correlation between bacterial virulence for humans and suckling mice and suggest that resistance to the bactericidal and opsonizing effects of normal human serum may be important factors governing that virulence.

Differential generation of chemiluminescence--detectable oxygen radicals by normal polymorphonuclear leukocytes challenged with sera from systemic lupus erythematosus and rheumatoid arthritis patients.

The generation of chemiluminescence (CL)-detectable oxygen radicals by normal human polymorphonuclear leukocytes (PMN) after challenging with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) sera is described. CL was measured in a luminol-dependent assay and referred to a standard obtained when performed immune complexes (Ic) (human tetanus toxoid-antitoxoid Ic resuspended in normal pooled serum) were tested on PMN. Normal sera gave rise to CL activity by PMN between 0% and 50% of the standard Ic (mean +/- standard error of the mean (SEM): 20.7 +/- 4.8). Sera from SLE and RA patients induced strikingly different biological effects on PMN. SLE sera generally induced a high CL-detectable generation of oxygen metabolites which may be causally related to the intense tissue damage (vasculitis) frequently observed in this disease. In contrast to SLE, RA sera induced a CL-detectable respiratory burst by PMN that was included in the normal range. Thus, the biological effects of these sera in terms of stimulation of toxic oxygen radical generation by phagocytes are quite different. This generation of oxygen radicals might reflect a different clearance of circulating Ic by PMN in SLE and RA disease.