Impairment of polymorphonuclear leukocyte function and metabolic control of diabetes.

OBJECTIVE--In this study, ingestion of Staphylococcus aureus and "bacteria killing" (BK) were measured to evaluate polymorphonuclear leukocyte (PMN) phagocytic functions and chemiluminescence response (CL) to phorbol-myristic acetate (PMA) as respiratory burst activity with regard to metabolic control parameters in diabetic patients. RESEARCH DESIGN AND METHODS--PMN phagocytic functions were assessed in 40 diabetic patients, all receiving insulin and in poor metabolic control, with 3H-thymidine-labeled Staphylococcus aureus in a modified radiometric assay. Bacteria killing was determined by pure-plate counting of surviving bacteria (colony-forming units [cfu]) and luminol-enhanced CL in response to PMA as a measure of respiratory burst. PMN function data were correlated to HbA1 as parameter of recent metabolic control. RESULTS--PMN of diabetic patients showed a significant reduction in Staphylococcus aureus (50.7 +/- 4.1%) and BK (29.4 +/- 4.2%) compared with healthy nondiabetic control subjects (76.6 +/- 4.6% and 16.3 +/- 3.1%, respectively, P less than 0.001), and PMN CL response was markedly reduced in diabetic patients also. Linear regression analysis showed a highly significant negative correlation of HbA1 versus Staphylococcus aureus (r = -0.67, P = 0.001) and a positive correlation for BK (r = 0.73, P less than 0.001). This was also true for CL, although this did not reach statistical significance (P = 0.06). CONCLUSIONS--The data obtained demonstrate impaired PMN phagocytic functions and CL response in diabetic patients. These findings suggest inhibitory effects of elevated glucose concentrations on PMNs, a possible role of protein glycosylation for impairing PMN function, thus contributing in part to altered host defense.     

Increased chemiluminescence of polymorphonuclear leucocytes from patients with progressive systemic sclerosis

Chemiluminescence (CL) of whole blood and isolated polymorphonuclear leucocytes (PMN) from patients with systemic sclerosis (SS) and from age and sex matched controls was measured. CL was induced by the addition of particles (zymosan, latex beads), or phorbol myristate acetate (PMA) or chemotactic peptide (FMLP). Whole blood CL (induced by PMA, zymosan or latex particles) was significantly greater in SS patients than in normal controls. Isolated PMN CL (induced by PMA, FMLP or latex particles) was also significantly greater in the SS patients compared with controls. Increased CL or PMN from patients with SS was mainly observed when luminol was used as amplifier (which detects hydrogen peroxide formation). In most cases, lucigenin-amplified CL of PMN from patients with SS (which detects the primary superoxide anion radical formation) did not differ from the controls. Sera from patients with SS significantly increased both spontaneous and induced CL of normal PMN. Enhanced excitability of PMN to phagocytosis-related stimuli may provide a mechanism for the (leucocyte-mediated) endothelial injury in SS.     

Hypertonic saline activation of p38 MAPK primes the PMN respiratory burst

Investigation of hypertonic saline (HTS) modulation of neutrophils (PMN) cytotoxic responses has generated seemingly contradictory results. Clinically relevant levels of HTS attenuate receptor-mediated p38 MAPK signaling, whereas higher levels activate p38 MAPK. Concurrently, HTS exerts a dose-dependent attenuation of the PMN respiratory burst, most notably at concentrations where p38 MAPK is activated. We hypothesized that HTS-mediated p38 MAPK activation augments the PMN respiratory burst on return to normotonicity. We found that although clinically relevant levels of HTS (Na+ > or = 200 mM) did not activate p38 MAPK, higher concentrations (Na+ > or = 300 mM) resulted in activation comparable with that after PAF stimulation. Transient stimulation with high levels of HTS primed the PMN respiratory burst in response to fMLP and PMA. This effect was attenuated by pretreatment with SB 203580, a p38 MAPK specific inhibitor. We conclude that severe osmotic shock primes the respiratory burst via p38 MAPK signaling, further supporting the role of this signaling cascade in PMN priming.     

Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis.     

Neutrophil-induced depletion of adenosine triphosphate in target cells: evidence for a hypochlorous acid-mediated process

Human neutrophils, incubated with phorbol myristate acetate (PMA), caused a rapid and substantial adenosine triphosphate (ATP) depletion in lymphoblastoid Daudi cells without producing lysis. Catalase (which destroys hydrogen peroxide), taurine and methionine (which scavenge hypochlorous acid), and chloride omission from the medium prevented the ATP fall. An ATP depletion comparable to that induced by neutrophils was observed by replacing neutrophils with an appropriate myeloperoxidase-H2O2-Cl- enzymatic system. Together, these data suggest that the neutrophil ATP depleting activity involves the myeloperoxidase-catalyzed transformation of H2O2 into HOCl. Moreover, the free H2O2 remaining in the neutrophil extracellular environment is ineffective. In fact, a comparable amount of enzymatically generated H2O2 did not cause Daudi cell ATP loss. A direct role for H2O2 in the neutrophil-induced Daudi cell ATP depletion was observed only under artificial conditions, that is, in the presence of the heme enzyme inhibitor azide, which prevented the HOCl production but dramatically augmented the extracellular H2O2 level. Similar levels of ATP depletion in Daudi cells were induced by amounts of reagent HOCl comparable to those generated by neutrophils. As the generated HOCl can rapidly react with a variety of neutrophil-derived nitrogenous compounds (primarily ammonia and taurine) to yield chloramines, these chlorinated oxidants might contribute to the neutrophil-mediated ATP depletion. Nevertheless, the main and well-characterized chloramines (ammonia-derived monochloramine, NH2Cl, and taurine monochloramine, TauNHCl) were devoid of ATP-depleting capacity. Thus, the results suggest that the neutrophil-induced ATP depletion in Daudi cells is HOCl-dependent, is not mediated by NH2Cl or TauNHCl, and could be promoted either by HOCl directly or by an unknown derivative oxidant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemiluminescence of Human and Canine Polymorphonuclear Leukocytes in the Absence of Phagocytosis

Polymorphonuclear leukocytes (PMNs) have increased oxidative metabolism during phagocytosis and emit light (chemiluminescence, CL) as a result of metabolic activation. The present study examined PMN CL in the absence of phagocytosis using sodium fluoride (NaF), a nonparticulate agent and known stimulator of cellular oxidative metabolism. Normal human and canine PMNs were assayed in a CL spectrometer which permitted continuous sample mixing and constant temperature regulation during CL measurement. PMNs treated with 20 mM NaF demonstrated maximum CL responses of 10,000-20,000 cpm above background, 13-17 min after addition of NaF at 37 degrees C. Temperature regulation of reaction mixtures was found to be a critical factor in assaying PMN CL responses to NaF, because a small decrease in temperature (i.e. 1.5 degrees C) substantially depressed and delayed the CL response. Superoxide anion production correlated closely with CL responses in NaF-treated human PMNs. CL responses were completely suppressed in the presence of the oxidative metabolic inhibitors, iodoacetamide, and N-ethylmalemide; and were partially suppressed in the presence of either superoxide dismutase or sodium azide.CL responses of NaF-treated PMNs were significantly lower than responses generated by PMNs phagocytizing opsonized yeast. When NaF was evaluated for its effect on light generation from a singlet oxygen dependent CL reaction, it was found that NaF did not quench singlet oxygen light. This study demonstrates that PMN CL can occur in the absence of phagocytosis, and it proposes that a nonphagocytic PMN CL assay may be useful in evaluating leukocyte metabolic defects.     

Role of hypochlorous acid and chloramines in the extracellular cytolysis by neutrophil polymorphonuclear leukocytes

The lysis of human red blood cells (HRBC) by neutrophil polymorphonuclear leukocytes (PMN), triggered with opsonized zymosan (OPZ) particles, was inhibited by azide, catalase, Cl- -free medium and amino acids indicating the involvement of myeloperoxidase (MPO), hydrogen peroxide (H2O2), Cl- ions and hypochlorous acid (HOCl) respectively. Thus, the cytolytic process depends on the following reaction: (Formula: see text). Because the oxidizing agent HOCl is also the precursor of the chloramines, a group of oxidants formed by the reaction between HOCl and PMN-derived ammonia (NH4+) or amines (R-NH2), the observed HRBC lysis can be theoretically due to HOCl and/or chloramines. Nevertheless, we found that PMN-mediated cytotoxicity occurs as an unidirectional process, being HRBC targets lysed and PMN unaffected. This finding indicates that the cytotoxin must be relatively more efficient against HRBC as compared with PMN. In fact, reagent HOCl (used at concentrations comparable to those generated by PMN) but not chloramines displayed such a type of property. Taken together, the data suggest that HRBC are killed by PMN-derived HOCl without the requirement for chloramines: this implies that NH4+ and R-NH2, released by PMN, act as down-modulators of the cytotoxic process, serving as HOCl trapping agents.     

Use of lipophilic probes of membrane potential to assess human neutrophil activation. Abnormality in chronic granulomatous disease.

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3''-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3''-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.     

Generation of nitrogen-chlorine oxidants by human phagocytes.

Human phagocytes can be triggered to generate large quantities of long-lived nitrogen-chlorine derivatives. This class of oxidants can be detected as early as 5 min after the addition of phorbol myristate acetate or opsonized zymosan particles. Unlike all other oxygen metabolites known to be generated by phagocytes, the nitrogen-chlorine compounds can be readily detected in cell supernatants 90 min after stimulation. The generation of these oxidants is linear with neutrophil concentration, favored at alkaline pH, and inhibited by supraphysiologic concentrations of iodide or bromide. The oxidants are hydrophilic in nature and have a half-life ranging from 5 h at 37 degrees C to greater than 100 h at 4 degrees C. Gel filtration chromatography of the accumulated nitrogen-chlorine derivatives revealed that the oxidants generated by neutrophils or monocytes are a complex mixture of products whose Mr range from 150-5,000. One-half of the nitrogen chlorine derivatives migrate as a single peak with an Mr of approximately 150. Amino acid analysis of this fraction identified the beta-amino acid, taurine, as the single nitrogenous compound present. Neutrophils triggered in the presence of serum albumin accumulated increased amounts of the nitrogen-chlorine derivatives while continuing to generate their endogenous low Mr oxidants. Quantitative analysis of the 36Cl incorporation revealed that the albumin molecule was chlorinated with the formation of both nitrogen-chlorine and carbon-chlorine bonds. We conclude that human phagocytes can chlorinate both endogenous and exogenous nitrogenous compounds at inflammatory sites to generate a heterogeneous mixture of nitrogen-chlorine derivatives. The ability of phagocytes to generate this class of long-lived oxidants whose hydrophilic characteristics restrict their localization to the extracellular space suggests that these species play an important role in modulating the inflammatory response.     

Enhanced phagocytic cell respiratory burst induced by spinal manipulation: potential role of substance P.

The effect of spinal manipulation on the respiratory burst of polymorphonuclear neutrophils (PMN) and monocytes from treated adults was measured by zymosan-stimulated chemiluminescence (CL). Peripheral blood was collected 15 min before and 15 min after treatment (sham manipulation, thoracic spine manipulation, or soft tissue manipulation), the cells were isolated, challenged with a standardized, opsonized luminol-containing suspension of zymosan, and monitored for CL. Plasma from two subsets of subjects was radioimmunoassayed for Substance P (SP). PMN were also preincubated with SP in vitro over the dose range 5 x 10(-12) M to 5 x 10(-8) M and the CL response monitored. The CL responses of both PMN and monocytes from subjects who received spinal manipulation were significantly higher after than before treatment, and significantly higher than the response in sham or soft-tissue treated subjects. Measurement of the force applied by sham and spinal manipulation suggested a force threshold for the enhancement of the CL response. Plasma levels of SP before and after treatment in sham treated subjects did not differ significantly; however, elevated plasma SP was observed in subjects after spinal manipulation. Preincubation of PMN with 1 x 10(-11) M, 5 x 10(-11) M or 1 x 10(-10) M SP in vitro primed PMN for an enhanced respiratory burst when the cells were subsequently challenged.     

Quantitative NBT reduction test with or without stimulation of peripheral polymorphonuclear cells from pregnant women

We performed the nitroblue tetrazolium (NBT) reduction test to investigate the function of polymorphonuclear cells (PMN) from prenatal, intrapartal, postpartal and nonpregnant women with or without stimulation by phorbol myristate acetate (PMN). The capacity for NBT reduction of PMN was significantly increased in pregnancy, followed by a further increases during labor without any stimulation. On the other hand, when stimulated with PMA, there were no differences between pregnancy and labor in the reactivities of PMNs, although they were still significantly higher than those of nonpregnant women. Kallikrein increased the activity of PMN in an in vitro study, suggesting that it may be associated with the change in PMN function in labor.     

Kinetics of chlorination of monochlorodimedone by myeloperoxidase

The phagocyte-derived enzyme myeloperoxidase has been recently implicated in the pathogenesis of atherosclerosis, because it catalyzes the reaction of hydrogen peroxide with chloride ions to give the highly toxic oxidant hypochlorous acid. The aim of this study was to determine the dependence of this reaction on the concentration of hydrogen peroxide and of the enzyme by means of the photometric monochlorodimedone assay. The initial rate of hypochlorous acid formation increased less than proportionally with increasing myeloperoxidase concentrations. Variation of the concentration of hydrogen peroxide had a biphasic effect, with an optimal concentration of hydrogen peroxide. Above this concentration enzyme destruction is apparently predominant. The progress curves of hypochlorous acid formation showed two distinct maxima. It was concluded that hypochlorous acid not only reacts with monochlorodimedone but also with the amino groups of myeloperoxidase to form intermediary chloramines that may further chlorinate monochlorodimedone. This was supported by the kinetics in the presence of the amino compound glycine, a competitive substrate for chlorination by hypochlorous acid. In the presence of high concentrations of glycine the progress curve rises continuously, yielding a greatly increased concentration of chlorinating species, either hypochlorous acid or chloramines. We concluded that glycine protects myeloperoxidase against hypochlorous acid-induced self-destruction.     

Re-evaluation of the phagocytic respiratory burst in the physiological or inflammatory state and in aging

We studied the differences in oxygen metabolite generation, using a chemiluminescence (CL) assay, in peripheral blood phagocytic cells from various donors including healthy young volunteers, patients with acute or chronic inflammation, pregnant women, and elderly persons. The CL response of polymorphonuclear leukocytes (PMN) after stimulation with serum-opsonized zymosan was increased in patients with acute inflammation due to infection and in pregnant women as compared with that in controls. Monocytes from those patients also showed a slight increase of the CL response. In contrast, CL of monocytes from patients with chronic inflammation (Crohn's disease patients) and elderly persons was significantly enhanced, whereas that of their PMN remained in the range of control values. The significance of these results was discussed.     

The influence of phorbol myristate acetate on the metabolism of neutrophils from carriers of sex-linked chronic granulomatous disease.

The present investigation has compared the influences of phorbol myristate acetate (PMA) and heat-killed bacteria (HKB) on oxygen consumption and glucose oxidation by polymorphonuclear leukocytes (PMN) from carriers of sex-linked chronic granulomatous disease (CGD). PMA or HKB caused neutrophils from CGD carriers, considered as a group, to consume oxygen and oxidize glucose-1-14C at rates that were statistically distinguishable from rates of normal controls and affected CGD hemizygotes. PMA at a final concentration of 1.0 micrograms per milliliter wass more effective and reproducible than a ratio of 50 HKB: 1 PMN in discriminating the partial abnormality of carrier PMN from normal PMN. Moreover, a deficiency in glucose oxidation by the PMN of one individual carrier was detectable using PMA stimulation when no defect was apparent with HKB. Results of the present investigation confirm and extend previous observations which have demonstrated the similarity in responses of PMA-treated normal and CGD PMN to the reactions produced by particulates under similar conditions.     

Chlorination of Taurine by Human Neutrophils: EVIDENCE FOR HYPOCHLOROUS ACID GENERATION

The model hydrogen peroxide-myeloperoxidase-chloride system is capable of generating the powerful oxidant hypochlorous acid, which can be quantitated by trapping the generated species with the β-amino acid, taurine. The resultant stable product, taurine chloramine, can be quantitated by its ability to oxidize the sulfhydryl compound, 5-thio-2-nitro-benzoic acid to the disulfide, 5,5′-dithiobis(2-nitroben-zoic acid) or to oxidize iodide to iodine. Using this system, purified myeloperoxidase in the presence of chloride and taurine converted stoichiometric quantities of hydrogen peroxide to taurine chloramine. Chloramine generation was absolutely dependent on hydrogen peroxide, myeloperoxidase, and chloride and could be inhibited by catalase, myeloperoxidase inhibitors, or chloride-free conditions. In the presence of taurine, intact human neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan particles generated a stable species capable of oxidizing 5-thio-2-nitrobenzoic acid or iodide. Resting cells did not form this species. The oxidant formed by the stimulated neutrophils was identified as taurine chloramine by both ultraviolet spectrophotometry and electrophoresis. Taurine chloramine formation by the neutrophil was dependent on the taurine concentration, time, and cell number. Neutrophil-dependent chloramine generation was inhibited by catalase, the myeloperoxidase inhibitors, azide, cyanide, or aminotriazole and by chloride-free conditions, but not by superoxide dismutase or hydroxyl radical scavengers. Thus, it appears that stimulated human neutrophils can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate taurine chloramine. Based on the demonstrated ability of the myeloperoxidase system to generate free hypochlorous acid we conclude that neutrophils chlorinate taurine by producing this powerful oxidant. The biologic reactivity and cytotoxic potential of hypochlorous acid and its chloramine derivatives suggest that these oxidants play an important role in the inflammatory response and host defense.     

Ratio of bacteria to polymorphonuclear neutrophils (PMNs) determines PMN fate

This study was designed to investigate how live Escherichia coli influence the fate of polymorphonuclear neutrophils (PMNs) in vitro. PMNs from 10 healthy volunteers were cocultured with or without live E. coli at different ratios. Heat-killed E. coli (Hk) were also added to PMNs at a ratio of 1:10. The PMNs were then analyzed by flow cytometry for cell death, reactive oxygen intermediates (ROI) production, and CD16 expression. Morphologic features were also assessed. PMN apoptosis was confirmed by DNA gel electrophoresis. Low doses of E. coli (PMN:E. coli ratios of 1:0.01 and 1:0.1) inhibited PMN apoptosis. In contrast, a high dose of E. coli (PMN:E. coli ratio of 1:10) increased PMN necrosis. ROI production was significantly greater at PMN:E. coli ratios of 1:10 and 1:10 (Hk) than at ratios of 1:0.01 and 1:0.1, or in PMNs cultured alone after a 15 or 30 minute coculture. CD16 expressions were significantly lower in PMNs cocultured with E. coli than in those cultured alone after a 4 or 12-h coculture. Tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-6 levels in cell-free supernatants were also measured. The mean percentages of apoptosis at PMN:E. coli ratios of 1:0.01 and 1:10 (Hk), and in PMNs cultured alone after a 12-h coculture showed significant inverse correlations with these cytokine levels in cell-free supernatants at 12 h. Our results demonstrate that low doses of live E. coli inhibits predominantly PMN apoptosis, whereas a high dose of E. coli increases necrosis. Augmented PMN bactericidal function, via inhibition of PMN cell death, may be beneficial for host defense against bacterial infection and/or sepsis.     

Functional effects of monoclonal antibodies (mAbs) against human polymorphonuclear (PMN) adherence antigens

Two mouse monoclonal antibodies (mABs), 25.31 raised against an subunit epitope of LFA1 antigen and Mol against an epitope of the complement receptor type 3 (CR3) were used for investigating their effects on human polymorphonuclear (PMN) functions. The two mABs have an inhibitory effect on PMN adherence. Furthermore, the PMN adherence strength depends upon the support and the adherence induces the capping process of these antigens. Other PMN functions dependent upon adherence were also altered by these two mAbs: random locomotion and that directed by formyl-methionyl-leucyl-phenylalanine (FMLP) or by activated serum, degranulation induced by opsonized or non opsonized zymosan but not by phorbol myristate acetate (PMA), iodination, K562 cell cytotoxicity. Luminol enhanced chemiluminescence of PMN was diminished by both mAbs when PMN were stimulated either by opsonized zymosan or by PMA. Our results confirm other workers' findings, and they are consistent with PMN functional abnormalities observed in children with congenital LFA1, Mol antigens defect.     

Differential effects of macrophage inflammatory chemokine-2 and keratinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 +/- 5 mmHg and were then administered anti-MIP- 2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringer's lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; microU/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (increased MPO, increased % esterase+ cells in tissue) into the lung and local tissue inflammation (increased IL-6/MIP-2, decreased IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.     

Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation.

Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.     

Differential function of polymorphonuclear leukocytes between in vivo and in vitro in tumor-bearing mice

In the present report, we compared activities of polymorphonuclear leukocytes (PMN) such as phagocytosis and bactericidal activity in vivo with those in vitro in sarcoma 180 (S 180)-bearing mice. Mice showed a remarkable leukocytosis and in increase in PMN fraction of peripheral blood leukocytes (PBL) after intraperitoneal injection of S 180 cells. Tumor-bearing mice infected with Escherichia coli (E. coli) intravenously and intraperitoneally showed an apparent delay in the clearance of bacteria compared to the non-tumor-bearing control mice. However, PBL of tumor-bearing mice showed a high phagocytic activity against beads and a high chemiluminescence (CL) activity. Dichlorofluorescein (DCFH) oxidation capacity of peripheral blood PMN in S 180-bearing mice after stimulation with phorbol myristate acetate (PMA) was about the same or a little stronger than that in control mice. On the contrary, serum and ascites of tumor-bearing mice strongly suppressed the phagocytic and bactericidal activities of casein-induced PMN against E. coli. During the early phase of E. coli infection, serum level of complement (C3) was not depressed in tumor-bearing hosts. From these results, it is concluded that leukocytosis and activation of functions of PMN in tumor-bearing mice were observed in vitro but they were not effective for the protection in the early phase of actual E. coli infection in vivo. The delay of in vivo clearance may be accounted for by a suppressive effect of serum components in tumor-bearing mice.