Evaluation of neutrophil activity and circulating immune complexes levels in diabetic patients

We have studied chemiluminescence produced by neutrophils stimulated by opsonized zymosan in insulin dependent (IDD) and non insulin dependent (NIDD) diabetic patients. Chemiluminescence was evaluated as the integral and maximum peak, total time and time to maximum peak of the response curve to opsonized zymosan. These values were then compared with circulating immune complexes (CIC) and glucose levels. Both IDD and NIDD patients had significantly higher values of chemiluminescence and CIC than normal controls. We also observed that patients who had the highest values of CIC and chemiluminescence levels were the ones with clinical microvascular complications.     

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.     

Effect of lithium on superoxide production and intracellular free calcium mobilization in elastin peptide (kappa-elastin) and FMLP stimulated human pmns. Effect of age

The effect of lithium pretreatment on superoxide anion production and intracellular free calcium levels was investigated in polymorphonuclear leukocytes (PMN) from middle-aged and old individuals after stimulation by elastin peptides or FMLP. K-elastin (KE) significantly stimulated the production of superoxide anion by PMNs from middle-aged subjects, while this stimulation decreased with age and was absent in PMNs of elderly arteriosclerotic patients. Li pretreatment slightly increased this stimulating effect of KE in PMNs from middle-aged subjects and elderly arteriosclerotic patiens, while slightly decreased in healthy elderly subjects. Moreover, Li was able to increase Superoxide anion production even in the absence of KE, but this effect decreased also in PMNs of healthy and arteriosclerotic elderly patients. FMLP significantly increased superoxide anion production in all age-groups, but this effect was further amplified by Li only in PMNs of middle-aged subjects. In aged individuals Li pretreatment slightly decreased the effect of FMLP and had no effect in arteriosclerotic patients. Ca-mobilization induced by KE was inhibited by Li pretreatement in each age group. This inhibition by Li was much weaker in FMLP-stimulated PMNs. Li pretreatment did however modify the shape of the Ca-transient curves in FMLP stimulated leukocytes suggesting a qualitative modification of ion channel regulation. No such shape change of Ca-transient curves was observed after KE stimulation of Li pretreated PMNs. It appears that the regulation of these two receptors is differently affected by Li treatement.     

The functions of circulatory polymorphonuclear leukocytes in diabetic patients with and without diabetic triopathy

We determined circulatory polymorphonuclear leukocytes (PMN) functions of superoxide anion production, adhesion and aggregation in 38 type 2 diabetic patients with and without diabetic triopathy. Tumor necrosis factor (TNF)-alpha-stimulated superoxide production and N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated aggregation in diabetic patients with triopathy were significantly greater than those in diabetics without triopathy. The more diabetic complications existed, the more TNF-alpha-stimulated superoxide was produced by PMN. These results suggest that the activated PMN contributes to a progression of diabetic triopathy in type 2 diabetic patients.     

Neutrophil dysfunctions, IL-8, and soluble L-selectin plasma levels in rapidly progressive versus adult and localized juvenile periodontitis: variations according to disease severity and microbial flora.

We used flow cytometry to analyze the expression of adhesion molecules and the oxidative burst of whole-blood polymorphonuclear neutrophils (PMN) from 26 patients with periodontitis. Three different clinical entities were studied: adult periodontitis (AP), localized juvenile periodontitis (LJP), and rapidly progressive periodontitis (RPP). Unstimulated PMN from the patients showed reduced Lewis x, sialyl-Lewis x, and L-selectin expression relative to those from healthy control subjects. These alterations were present whatever the severity of periodontal disease. However, PMN from RPP patients showed increased basal H2O2 production and decreased L-selectin shedding. These latter impairments, which correlated with increased IL-8 plasma levels, could contribute to initial vascular damage. In addition, decreased IL-8 priming of H2O2 production by PMN from RPP patients could account for a lower bactericidal capacity of PMN, leading to the large number of bacteria in the subgingival region of RPP patients. Soluble L-selectin plasma levels were also decreased in the RPP group, indicating more severe or diffuse endothelial damage. These abnormalities were not found in the patients with less destructive forms of periodontitis (AP and LJP). Porphyromonas gingivalis, a bacterial pathogen known to increase IL-8 production by PMN, was found in the periodontal pockets of RPP patients only. These results show links among PMN abnormalities, the clinical form of periodontitis, and the gingival bacterial flora.     

Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Effects of in vitro and in vivo supplementation with zinc on superoxide anion production in leukocytes

The effects of zinc on the rate of production of bactericidal O2- of polymorphonuclear leukocytes (PMN) in response to three different types of stimulating agents (serum-treated zymosan (STZ), Con A, and myristate) were studied. The percentage reduction of O2- production of PMN stimulated by STZ, Con A, and myristate were all reduced in response to Zn, irregardless of whether Zn was added to the reaction mixture immediately before SZT addition or following a prior 20 min. incubation of PMN in the presence of Zn. However, when Zn was introduced intraperitonially into guinea pigs before the collection of PMN from the animal, zinc treatment produced inhibition only in STZ-activated PMN; it produced no effect in O2- production of PMN stimulated by myristate, and it further augmented the O2- production stimulated by Con A.     

Phagocytosis and neutrophil bactericidal capacity in patients with uremia

The phagocytic activity and bactericidal capacity of polymorphonuclear neutrophils (PMN) were evaluated in patients with advanced chronic renal failure. The studies were made in patients undergoing hemodialysis, maintenance peritoneal dialysis as well as in nondialysed patients. Evaluations were carried out by using of the recently described fluorochrome microassay which enabled these parameters to be estimate independently. The phagocytic activity was seriously diminished in nondialysed patients, whereas it was similar to controls in those hemodialysed and undergoing peritoneal dialysis patients. In all evaluated groups of patients the bactericidal capacity was significantly reduced. The lowest values could always be observed in nondialysed patients. The decrease of bactericidal capacity was significantly more evident in patients undergoing peritoneal dialysis as compared with those hemodialysed. The obtained results confirm some previous reports suggesting the impairment of PMN function in uremic patients. This results in their increased susceptibility to infection. They also reveal the existence of a close relationship between the extent of observed dysfunctions and the management applied.     

Superoxide anion generation, erythrocytes superoxide dismutase activity, and lipid peroxidation during hemoperfusion and hemodialysis in chronic uremic patients

In 11 chronic uremic patients superoxide anion generation in whole blood, both without and with opsonized zymosan stimulation, was lower than that in 11 healthy controls, while erythrocyte superoxide dismutase (SOD-1) activity and erythrocyte and plasma malonyldialdehyde (MDA) concentrations were elevated. During hemoperfusion (HP) and hemodialysis (HD) superoxide anion generation transiently significantly increased. Changes in the erythrocyte SOD-1 activity and plasma and erythrocyte MDA concentrations during HP suggested that this procedure exerted beneficial effects on lipid peroxidation. On the other hand, during HD erythrocyte membrane lipid peroxidation seemed to be enhanced even further; this phenomenon took place mainly within the dialyzer and a decrease in the erythrocyte SOD-1 activity seemed to be one of the contributing factors. Results of in vitro experiments with cross-incubation of erythrocytes and blood plasma and incubation of whole blood with cuprophan membrane suggest existence of an SOD-1 activator in the uremic blood plasma, which is possibly eliminated during HD.     

Production of superoxide and nitric oxide by granulocytes in non-insulin-dependent diabetic patients with and without diabetic nephropathy

Production of the superoxide radical anion O2-. and the nitric oxide radical NO-. by granulocytes was studied in 14 patients with type 2 diabetes without nephropathy, 21 patients with type 2 diabetes and diabetic nephropathy, and 19 healthy subjects, both without and after stimulation with opsonized zymosan. O2-. production by both resting and stimulated granulocytes was increased in type 2 diabetes patients without nephropathy but decreased in type 2 diabetes patients with nephropathy, compared with healthy subjects. NO. generation was highly augmented in type 2 diabetes patients without nephropathy by both resting and stimulated cells; values for type 2 diabetes patients with nephropathy were intermediate between the type 2 diabetes patients without nephropathy and the healthy subjects. These data point to granulocytes as one of possible sources of oxidative stress in type 2 diabetes.     

Free radical generation, lipid peroxidation and essential fatty acids in uncontrolled essential hypertension

Vascular endothelium produces prostacyclin (PG12) and endothelium-derived vascular relaxing factor (EDRF), which are potent vasodilators and hence, may have a role in the regulation of blood pressure. Both PG12 and EDRF are readily degraded by free radicals, especially superoxide anion. Hence, we studied free radical generation and lipid peroxidation in patients with uncontrolled essential hypertension. It was observed that superoxide anion and hydrogen peroxide production by polymorphonuclear leukocytes (PMN) and the levels of lipid peroxides (measured by thiobarbituric acid assay) were higher in uncontrolled hypertensives compared to controls. Both free radical generation and the levels of lipid peroxides reverted to normal values when assayed after the control of hypertension. The calcium antagonist, verapamil, and beta-1 blocker, metoprolol, at the doses used inhibited free radical generation by phorbolmyristate acetate-stimulated PMNs. On the other hand, angiotensin II augmented free radical generation in normal PMN. In addition, it was also observed that both linoleic acid and arachidonic acid levels are low in the plasma of patients with hypertension compared to controls. These results suggest that increase in free radical generation by PMN and alterations in the plasma concentrations of essential fatty acids are closely associated with uncontrolled hypertension.     

Priming of superoxide anion in polymorphonuclear neutrophils by brain natriuretic peptide.

In acute coronary syndromes such as unstable angina and myocardial infarction, serum concentration of brain natriuretic peptide, a cardiac hormone with potent vasodilatatory, natriuretic and diuretic activities, is elevated. Little is known about the effect of elevated BNP plasma concentration on free radical-mediated tissue damage in these states. We investigated the influence of human BNP 32 and its fragment BNP 7-32 on the production of superoxide anion by PMN, a major cause for myocardial damage. Although BNP showed itself no stimulatory potential on superoxide anion release in PMN, it enhanced significantly the stimulatory potential of cell stimuli such as fMLP or phorbol 12-myristate 13-acetate (PMA) in PMN. Thus our data show that the cardiac-derived hormone BNP influences an important function of PMN. This 'priming' effect of BNP on PMN may contribute to the tissue damage occuring during acute coronary syndromes.     

Phagocytosis of immunoglobulin G and C3-bound human sperm by human polymorphonuclear leukocytes is not associated with the release of oxidative radicals.

Antisperm antibody (ASA)- and complement (C)-mediated immune injury to human sperm is thought to be caused in part by phagocytic neutrophils. To investigate this process, we co-cultured purified human polymorphonuclear leukocytes (PMN) with swim-up sperm in the presence of ASA-positive and ASA-negative sera and assayed for PMN respiratory burst activity, monitored by the release of superoxide anion (O2-) and hydrogen peroxide (H2O2). Phorbol myristate acetate (PMA) and opsonized zymosan were used as positive controls. Phagocytosis of ASA-positive and C-bound sperm by PMN did not enhance O2- production when compared to incubation of sperm with ASA-negative sera. Phagocytosis of ASA-positive and C-bound sperm also resulted in minimal release of H2O2 when compared with ASA-positive and C-negative sperm that were not phagocytosed. In contrast, PMN were maximally stimulated to release O2- in response to either opsonized zymosan or PMA. The kinetics of PMA-induced O2- release was unaffected by the presence of ASA-positive and C-bound sperm. Cytocentrifuge preparations of PMN incubated with ASA-positive and C-bound sperm revealed limited O2- release at the site of PMN/sperm contact. These results indicated that 1) phagocytosis of motile sperm by PMN requires the binding of both ASA and C to the sperm surface; 2) phagocytosis of ASA-positive and C-positive sperm by PMN fails to release reactive oxygen species; and 3) metabolic processes associated with PMN respiratory burst activity may not be coupled to the ingestion of ASA-positive and C-bound sperm.     

Obstetric history of diabetics: its relevance to the aetiology of diabetes.

The birth weights of infants born to patients with insulin-dependent diabetes (IDD) and insulin-independent diabetes (IID) before the disease was diagnosed were compared. An appreciable excess of infants above the 90th centile for weight was found, the proportions being 27% of infants born to mothers with IDD and 30% of those born to mothers with IID. These findings suggest that many patients with both types of diabetes have a prolonged period of metabolic abnormality before overt symptoms of diabetes arise and that the apparent acute onset of the disease in patients who are insulin dependent is illusory.     

Studies on the molecular mechanisms of human neutrophil Fc receptor-mediated phagocytosis. Evidence that a distinct pathway for activation of the respiratory burst results in reactive oxygen metabolite-dependent amplification of ingestion

Human neutrophils (PMN) possess at least two distinct mechanisms for the ingestion of IgG-opsonized pathogens; one is independent of and the other is dependent on products of the respiratory burst. Oxidant-mediated ingestion is not induced by exposure to the IgG-opsonized target but requires additional stimulation by phorbol esters or cytokines. The purpose of the present work is to elucidate the signal transduction pathways underlying these two distinct phagocytic mechanisms. Both phorbol ester- and cytokine-stimulated ingestion of IgG-opsonized targets and superoxide anion production were inhibited by the protein kinase C (PKC) inhibitors TFP and H7. In contrast, neither phagocytosis nor superoxide anion generation induced by stimulation with IgG-opsonized targets alone was affected by either of these inhibitors, even when IgG opsonization was increased to generate equal levels of ingestion and superoxide anion as that observed with cytokine stimulation. Moreover, TNF-alpha and IgG-opsonized target stimulation of PMN showed marked synergy in translocation of PKC activity from the cytosol to the plasma membrane. These data indicate that a pathway for activation of the respiratory burst which is dependent on protein kinase C is involved in oxidant-mediated amplification of ingestion. Cytokine stimulation of PMN not only augments IgG-dependent ingestion and generation of superoxide anion but also changes the signaling pathway for these two IgG-dependent functions from PKC-independent to PKC-dependent. In this regard, cytokine stimulation differentiates two pathways for activation of PMN by IgG.     

Platelet factor 4 (PF4) and heparin released platelet factor 4 (HR-PF4) in diabetes mellitus. Effect of the duration of the disease

Several investigators have reported an altered platelet function in diabetes mellitus as measured by elevated levels of platelet specific proteins platelet factor 4 (PF4) and B-thromboglobulin (BTG). We studied 20 insulin dependent (IDD), 20 non insulin dependent (NIDD) diabetic males without overt clinical symptoms of cardiovascular disorders and 30 normal controls. We evaluated PF4, BTG and heparin released platelet factor 4 (HR-PF4) as measured 2.5 minutes after a bolus injection of 5,000 I.U. of a commercial mucous heparin. The patients showed normal levels of both PF4 and BTG. Furthermore HR-PF4 failed to show statistically significant variation between patients and controls. However when the diabetics were divided on the basis of the duration of the disease, the IDD had an increased HR-PF4 mean level and the trend became statistically significant when diabetes existed more than 17 years (patients HR-PF4 149.1 ng/ml, range 17.3-194; controls HR-PF4 110.9 ng/ml range 50-160, less than p less than 0.05). NIDD failed to reveal the same pattern. Although the significance of HR-PF4 is unknown, insulin dependent diabetes mellitus after many years could cause a potentially dangerous, silent vascular damage with enhanced platelet vessel wall interaction as measured by an elevated HR-PF4.     

Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.     

Consumption of complement and activation of human neutrophils by an artificial immune complex: polyacrylic acid-IgG-polymer

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 micrograms/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.     

Oxygen-derived free radicals producing activity and survival of activated polymorphonuclear leukocytes

Summary Activation of polymorphonuclear (PMN) leukocytes is known to generate oxygen free radicals (OFR). However the fate of activated PMN leukocytes is not known. We investigated the OFR producing (chemiluminescence) activity and the survival of the activated PMN leukocytes. The study was divided into two groups. Group I, In vivo study (n = 7): zymosan (8.4 mg/kg) was administered intravenously in the anesthetized dogs and the blood samples were collected before and after 5, 15, 30, 60 and 120 min of zymosan administration. This group represents the in vivo pre-stimulated PMN leukocytes; Group II, In vitro study (n = 7): the blood were collected from dogs and further divided into two groups. Group A (n = 7): non-stimulated, without any added zymosan and group B (n = 7): zymosan was added to stimulate PMN leukocytes. Blood samples from group A and B were also collected at various time intervals similar to in vivo studies. Oxygen free radical producing activity of PMN leukocytes was monitored by measuring luminoldependent chemiluminescence (CL). Opsonized zymosan was used to activate PMN leukocytes. The studies in which the PMN leukocytes were stimulated in in vivo, both oxygen derived free radicals and superoxide dismutase (SOD) inhibitable oxygen free radical CL decreased significantly for 60 min and tended to reach thereafter to the pre-stimulated values. The resting chemiluminescence (chemiluminescence without zymosan stimulation in the assay medium) increased significantly for 15 min reaching to pre-stimulated values at 30 min and thereafter. In in vitro studies, oxygen derived free radicals CL of pre-stimulated PMN leukocytes (Group B) was depressed for the whole duration of investigation while SOD inhibitable CL was depressed for only 60 min. There was approximately a two-fold increase in the resting CL within 5 min of PMN leukocyte activation and it remained high for the whole duration of study. The chemiluminescence of non-stimulated PMN leukocytes in vitro (group A) remained practically normal throughout the period of observation. In in vivo studies, total white blood cells (WBC) and PMN leukocyte counts decreased initially and tended to approach towards pre-stimulated values at the end of the protocol. There were no changes in these counts in in vitro studies. These results indicate that the capacity to generate OFR is decreased in the in vivo and in vitro pre-stimulated PMN leukocytes. However this activity recovers with time. This study also suggests that the activated PMN leukocytes are not destroyed.