Simultaneous Intraperitoneal Administration of Ok-432 and Serum Enhances Superoxide Generation from Migrated Polymorphonuclear Leukocytes, with Special Emphasis on the Role of Complements

Superoxide and its derived active oxygen species are responsible for the polymorphonuclear leukocyte (PMN)-mediated tumoricidal activity which is typically shown in the intraperitoneal administration of OK-432, a biological response modifier, for cancer ascites. We examined the effects of intraperitoneal administration of OK-432 with or without syngeneic serum on superoxide generation from PMNs which migrated in the peritoneal cavity using the new method of Cypridina luciferin analog-dependent chemiluminescence for the detection of superoxide. PMNs harvested from rat peritoneal cavity 6 h after the intraperitoneal administration of OK-432 (0.25KE/kg, or 2.5KE/kg) generated larger amounts of superoxide than those harvested after intraperitoneal injection of oyster glycogen (75mg/kg) when stimulated by opsonized zymosan or phorbol myristate acetate. Simultaneous intraperitoneal administration of OK-432 and syngeneic serum induced a greater increase in PMN superoxide generation than OK-432 alone, which was reversed by a complement activation inhibitor (MX-1). Simultaneous injection of OK-432 and heat-inactivated syngeneic serum did not exhibit a significant increase in PMN superoxide generation as compared with OK-432 alone. These results provide pharmacological evidence to the satisfactory therapeutic effects of the intraperitoneal administration of OK-432 with or without serum for patients with cancer ascites, and indicate that complements, in particular C5a, are involved in this enhanced PMN-derived superoxide generation induced by the simultaneous injection of OK-432 and serum.     

PSK and OK-432-Induced Immunomodulation of Inducible Nitric Oxide (NO) Synthase Gene Expression in Mouse Peritoneal Polymorphonuclear Leukocytes and NO-Mediated Cytotoxicity

Abstract

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

OK-432-induced cytokines production by peripheral blood mononuclear cells]

Cytokines production by OK-432-stimulated peripheral blood mononuclear cells (PBMC) were measured to investigate the in vitro function of macrophages (M phi) and lymphocytes. PBMC (1 x 10(6) cells/ml) were cultured with OK-432 (0.05 KE/ml) for 72 hr at 37 degrees C under 5% CO2, then interleukin 1 beta (IL-1 beta), interleukin 2 (IL-2) and soluble IL-2 receptor (sIL-2R) levels in the culture supernatants were measured by ELISA. While there was no significant differences of IL-1 beta production between patients with chronic active hepatitis type B (CAH-B) and controls, sIL-2R production (335 +/- 219 U/ml, mean +/- SD) was significantly decreased (p < 0.001) in patients with CAH-B. On the other hand, in pregnant women, production of both IL-1 beta (6.3 +/- 3.9 ng/ml, p < 0.01) and sIL-2R (300 +/- 169 U/ml, p < 0.001) were significantly lower than those in controls (13.5 +/- 3.8 ng/ml, 969 +/- 154 U/ml). These results suggest that the expression of IL-2R alpha on lymphocytes membrane is suppressed in patients with CAH-B, and that decreased M phi function is present in pregnant women.     

Mechanism of Induction of Endogenous Tumor Necrosis Factor in Ascites of Ovarian Cancer Patients by OK-432, A Streptococcal Preparation

In four ovarian cancer patients with malignant ascites, 10 KE of OK-432 was intraperitoneally administered four times every other day for priming, and 40 KE of OK-432 in a single dose by the same route on day 13 for triggering. The changes in peripheral blood monocytes and intraperitoneal macrophages and the production of tumor necrosis factor (TNF) by peripheral blood mononuclear cells (PBMC) and ascitic lymphoid cells (ALC) were examined. In two of the four patients in whom TNF was induced in the ascites, the TNF production by PBMC and ALC was noted during priming, and after triggering, an increase in both the number of intraperitoneal macrophages and the TNF production by ALC was noted. In two other patients in whom TNF was not detected in the ascites, the ratio of intraperitoneal macrophages to ALC did not change throughout the whole period, and the TNF production by ALC was not augmented. These findings suggest that the priming administration of OK-432 can induce both intraperitoneal macrophages and peripheral blood monocytes into a primed state, and the triggering administration of OK-432 can increase the number of intraperitoneal OK-432-primed macrophages and induce TNF release from these cells.     

Oral administration of a streptococcal agent OK-432 activates peritoneal macrophages in mice

The effect of orally administered OK-432, a streptococcal preparation, on the function of peritoneal macrophages in mice was examined. The administration of OK-432 orally (1 KE or 2 KE, four times every three days) did not affect the numbers of both total peritoneal cells and macrophages recovered five days after the final administration. However, the macrophages exhibited an increase in their spreading ability. Other functions of the peritoneal macrophages including lysosomal enzyme activity, phagocytic activity and interleukin 1 (IL-1) production were also enhanced significantly by the oral administration of OK-432 (1 KE or 2 KE). The production of H2O2, however, was not affected by the same treatment with OK-432. The activation of peritoneal macrophages by orally administered OK-432 reported here may contribute to expansion of the clinical application of this drug.     

Production of interleukin 6 by human spleen cells stimulated with streptococcal preparation OK-432

The streptococcal preparation OK-432 was tested for the ability to stimulate human spleen leukocytes (SPL) for generation of interleukin 6 (IL-6). When SPL were cultured with OK-432 for 24 h in serum-free T medium, the cell-free supernatant induced production of IgM in the SKW6.CL-4 and IgG in the CESS human B cell line, while no such activity was detected in unstimulated SPL culture. The activity was neutralized by treatment with antiserum directed against B cell stimulatory factor 2 (BSF-2). An optimum production of BSF-2 was observed when SPL were stimulated with 10 micrograms/ml of OK-432. The culture supernatant also induced proliferation of IL-6-dependent murine hybridoma MH-60.BSF2 (hybridoma growth factor; HGF). It is thus evident that the molecule produced by OK-432-activated human SPL is BSF-2/HGF/IL-6. These results indicate that the antitumor agent OK-432 stimulates human spleen cells to produce IL-6.     

Enhancement of natural killer cell activity in human immunodeficiency virus-infected subjects by in vitro treatment with biologic response modifier OK-432.

A decrease in natural killer (NK) cell function has been related to the progression of human immunodeficiency virus (HIV) infection. In the present study, we assessed the ability of a streptococcus-derived biologic response modifier, OK-432, to augment NK lysis of uninfected K562 and U937 cells and HIV-infected U937 cells by peripheral blood mononuclear cells (PBMC) from HIV-seropositive homosexual men. Optimal two- to fourfold increases in lysis of the three targets were observed after pretreatment of PBMC from HIV-negative subjects for 4 h with 2 micrograms of OK-432 per ml. This effect was related primarily to gamma interferon (IFN-gamma) production induced by OK-432 and was not linked to production of tumor necrosis factors alpha and beta or to monocytes in the cultures. The enhancing effect of OK-432 on NK cell function was diminished but still evident in PBMC from subjects with relatively early-phase (< 3-year) HIV infection and high CD4+ cell counts and was lower in subjects with longer-term HIV infection (> 3 years), in association with reduced production of IFN-gamma. Augmentation of NK cell activity in HIV-infected men by OK-432 was comparable to that induced by treatment of cells with 1,000 U of IFN-alpha or interleukin 2 per ml. The data suggest that the NK cell-enhancing effects of OK-432 are at least in part mediated by IFN-gamma and that OK-432 may be effective in treatment of patients with early-phase HIV infection.     

Augmentation of interleukin 1 and interleukin 2 production by OK-432

Intraperitoneal (i.p.) administration of OK-432 augmented both interleukin 1 (IL-1) and interleukin 2 (IL-2) production to the rechallenge of OK-432 in vitro. Peritoneal exudate cells (PEC) of mice 8 days after i.p. injection with OK-432 (1 KE/mouse) showed maximum IL-1 production to the restimulation with OK-432 in vitro. OK-432-induced IL-1 was consisted of three molecular weight species (two major peaks: 85 K and 15 K daltons and one minor peak: 67 K daltons) on Sephadex G-100 chromatography. Splenocytes of mice 4 days after i.p. injection with OK-432 (1 KE/mouse) demonstrated maximum IL-2 production to the in vitro rechallenge of OK-432, however, in vivo OK-432 administration failed to enhance ConA-induced IL-2 production in vitro. From gel filtration analysis, OK-432 induced IL-2 had an unique molecular weight (approximately 70 K daltons). From these results, OK-432-induced augmentation of cellular immunity against tumor cells might be due to the activation of so-called lymphokine cascade reaction mediated by IL-1 and IL-2.     

Synergy between tumour necrosis factor α and granulocyte-macrophage colony-stimulating factor in neutrophil stimulation

We have examined the interaction between the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor α (TNFα) on polymorphonuclear leukocyte (PMN) function and on PMN responses to further stimulation with formyl Met Leu Phe (fMLP). Incubation of PMN with TNFα (0.3 nM) and, to a lesser extent, with GM-CSF (1 nM) directly stimulated superoxide anion (O ⁻₂ ) generation and increased the response to subsequent stimulation of PMN by fMLP (100 nM). However, the combination of GM-CSF and TNFα did not result in increased O ⁻₂ generation and there was no synergistic effect of the combination of these cytokines on the priming of fMLP-induced O ⁻₂ generation. The combination of TNFα and GM-CSF did result in a striking synergism in the stimulation of PAF generation, and, whereas neither stimulus alone resulted in detectable PAF release, the combination elicited the release of significant levels of PAF. The observation of significant PAF release from PMN exposed to TNFα and GM-CSF indicates that overt neutrophil stimulation with phagocytic or soluble stimuli may not be required for expression of at least some of the PMN pro-inflammatory capacity.

     

PSK and OK-432-induced immunomodulation of inducible nitric oxide (NO) synthase gene expression in mouse peritoneal polymorphonuclear leukocytes and NO-mediated cytotoxicity

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

Endothelin-1 does not prime polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites

The in vitro effect of endothelin-1 (ET-1) on the capacity of polymorphonuclear leukocytes (PMNLs) to generate reactive oxygen species (ROS) was investigated. Human PMNLs were separated from healthy volunteers and preincubated for 10 min. at 37°C with varying concentrations (10^–7–10^–12 M) of ET-1. After subsequent stimulation with FMLP (10^–7 M) or opsonized zymosan (0.5 mg/ ml) the intra- and extracellular generation of ROS was assessed by luminol-amplified chemiluminescence, superoxide radical (·O ₂ ^– ) and hydrogen peroxide (H₂O₂) production.Results: ET-1 alone failed to stimulate ROS generation. Neither the capacity for extracellular generation of oxygen metabolites nor the production of ROS with an intracellular origin was changed after preincubation of PMNLs with ET-1. ET-1 did not cause a shift of the ·O ₂ ^– /H₂O₂ production ratio after stimulation of PMNLs with FMLP. These findings suggest that ET-1 in vitro does not prime human PMNLs for enhanced production of ROS.     

OK-432 treatment increases matrix metalloproteinase-9 production and improves dimethylnitrosamine-induced liver cirrhosis in rats

Kupffer cells are major matrix metalloproteinase-producing cells in the liver. The production of metalloproteinases in Kupffer cells may contribute to the improvement of liver fibrosis inducing liver cirrhosis. In this study, we examined the effect of the OK-432 (a biological response modifier) on the dimethylnitrosamine-induced liver cirrhosis in rats. Dimethylnitrosamine (10 microg/ml) was injected intraperitoneally into 20 male Wistar rats 3x/week for 4 weeks. For the subsequent 4 weeks, the animals were injected with saline (1 ml, 1x/week) (Group I, n=10) or OK-432 (1 Klinishe Einheit, 1x/week) (Group II, n=10). The control rats were injected with 1 ml saline for the initial 4 weeks and subsequent 4 weeks (Group III, n=10). The degree of hepatic fibrosis, the immunolocalization of type IV collagen, hyaluronic acid, and alpha-smooth muscle actin, and the mRNA expression by Northern blotting and the activity by gelatin zymography of metalloproteinase-9 were evaluated. Serum aminotransferase, hyaluronic acid, interleukin-1beta and tumor necrosis factor-alpha levels were measured. The deposition of á-smooth muscle actin and extracellular matrix containing type IV collagen and hyaluronic acid was markedly suppressed by OK-432. The mRNA expression and the activity of metalloproteinase-9 were markedly increased by OK-432. The serum aminotransferase and hyaluronic acid levels were decreased by OK-432. The serum interleukin-1 and tumor necrosis factor-alpha values were lower than the detectable limit in all samples from all three groups. These results indicate that OK-432 increased the production of metalloproteinase-9 and improved the rat dimethylnitrosamine-induced liver cirrhosis. OK-432 is suggested to be useful for the treatment of liver cirrhosis.     

Simultaneous measurement of adherence and chemiluminescence by polymorphonuclear leukocytes

Oxygen radical release from adhering polymorphonuclear leukocytes (PMN) has been implicated as an important feature of many vascular diseases. We developed a technique by which adherence and production of O₂ radicals by PMN can be measured simultaneously. The technique combines the conventional nylon fiber assay for measuring adherence of PMN with concurrent scintillation counter measurement of chemiluminescence(CL) to assess O₂ radical production by PMN. We found that adherence of PMN to nylon fiber is associated with increases in CL. Moreover, increases in CL appear to be dependent on generation of O₂ radicals from PMN since they are not seen with PMN from a patient with chronic granulomatous disease (CGD) or in the presence of O₂ radical scavengers, superoxide dismutase, or catalase. Furthermore, agents which increase the adherence of PMN to nylon fiber are associated with increases in CL. Use of this approach may facilitate simultaneous evaluation of adherence and O₂ radical generation by PMN.This work was supported by the National Institutes of Health, the Council for Tobacco Research, the American Lung Association, the American Heart Association, the Kroc, Swan, Hill, Kleberg, and R. J. Reynolds Foundations. Dr. Repine was an Established Investigator of the American Heart Association during the conduct of this rsearch. Dr. Clifford is a recipient of the Parker B. Francis Fellowship Award.     

Therapeutic Effect of Ok-432 Induced Endogenous Tnf on Tumor Bearing Mice and Cancer Patients

The therapeutic effect of OK-432 induced endogenous TNF on tumor bearing mice and cancer patients was investigated.

OK-432 (10 KE/mouse) was administered intraperitoneally to Balb/c mice 7 days prior to the transplantation of Meth A cells (1×10⁶/mouse) into the abdominal cavity. And at day 1 of tumor inoculation, 1 KE/mouse of OK-432 was administered intraperitoneally.

The significant prolongation of life span was observed in these mice.

On the basis of these observation, therapeutic effect of endogenous TNF on cancer patients was clinically evaluated. OK-432 was administered intraperitoneally or intrapleurally to cancer patients with peritonitis carcinomatosa or pleuritis carcinomatosa 4 times (10KE each) every other day and 50KE of OK-432 was readministered with the interval of 7 days.

An appreciable activity of TNF was detected in peritoneal fluids or pleural effusion, and the significant decreasing of these fluids was observed. It is therefore concluded that these therapeutic approach may well be taken into account in treatment of cancer.     

In vitro effects of leukotriene B4 (LTB4) on canine PMN effector function(s)

The canine has become an accepted research model for the examination of a number of human clinical conditions. Despite it's status as a research model, little is known regarding the peripheral effects of inflammatory mediator substances. Products of arachidonic acid metabolism (leukotrienes) are reported capable of altering leukocyte functions. Because of the emerging importance of the canine research model and leukotrienes we examined the effects of leukotriene B₄ (LTB₄) on severalin vitro functions of isolated canine peripheral polymorphonuclear leukocytes (PMN). Changes in forward angle light scatter properties of the cells were used as one measure of PMN activation. Other functional changes examined following LTB₄ pretreatment included chemotactic capability, the electrophysiological state of the cell plasma membrane, and the metabolic oxidative response (i. e. H₂O₂ production). Random cellular movement of PMNs increased by 120% and 72% following preincubation with 10^?7 and 10^?9 M LTB₄, respectively. LTB₄ between 10^?7 and 10^?13 M did not significantly alter cellular resting membrane potential. Between 10^?7 and 10^?9 M LTB₄ elicited significant levels of cellular H₂O₂ production. Although significant, H₂O₂ production was <40% that induced by phorbol myristate acetate (PMA). In numerous respects, caninein vitro PMN responses parallel previous reports of human cell function(s) in the presence of inflammatory mediators and may represent an attractive alternative for investigation of PMN dysfunctions.     

Influence of Low Molecular Weight Heparin (Certoparin) and Unfractionated Heparin on the Release of Cytokines from Human Leukocytes

We analyzed the influence of heparins (unfractionated heparin, UFH and low molecular weight heparin certoparin) on the generation of IL-1ra, IL-6, IL-10, and IL-12p40 and from leukocyte fractions in vitro. Polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from 16 different healthy donors were isolated and adjusted to 1 × 10⁶ cells/ml supplemented RPMI 1640. Leukocyte fractions were differentially stimulated (PMN with 1 g and 5 g LPS, PBMC with 10 ng TSST-1 or 2 g ConA) in the presence or absence of heparins (1 U/ml, 2 U/ml, and 4 U/ml) for 24 h at 37°C. Cytokine release was analyzed by ELISA. Certoparin but not UFH led to a dose-dependent increase in IL-6 from non-stimulated PBMC. In contrast, the release of IL-1ra, IL-10, and IL-12p40 was not modulated by heparins in a dose-dependent fashion. Increases in these cytokines occurred only as single incidents at intermediate heparin levels. An influence of the heparins on the apoptosis of PMN (measured as DNA-fragmentation in non-stimulated or LPS-stimulated cell-fractions) was not observed.     

Bacterial lipopolysaccharide enhances polymorphonuclear leukocyte function independent of changes in intracellular calcium

Bacterial lipopolysaccharide (LPS) enhanced expression of C3bi receptors (CR3), phagocytosis of opsonized bacteria, and subsequent hydrogen peroxide (H₂O₂) production by human polymorphonuclear leukocytes (PMNs). The role of changes in intracellular calcium concentration ([Ca²⁺]_i) in LPS-induced priming was examined by determining the effect of modulators of intracellular calcium on enhanced PMN function, determining the ability of calcium ionophores to reproduce the effects of LPS, and measuring PMN [Ca²⁺]_i following addition of LPS. Inhibition of intracellular calcium-dependent processes with TMB-8 or quin-2 blocked all three measures of LPS-induced priming. LPS did not stimulate an increase in [Ca²⁺]_i, and calcium ionophores failed to reproduce the effect of LPS. Maintenance of [Ca²⁺ _i is necessary for LPS priming, but an increase in [Ca²⁺]_i is not a component of the signal transduction pathway leading to PMN priming by LPS.     

Efficacy of in vitro sensitized cells generated by in vivo priming with OK-432 for adoptive immunotherapy of the poorly immunogenic B 16-BL6 melanoma

We investigated the efficacy of the streptococcal preparation OK-432 as an adjuvant for in vivo priming in induction of sensitized cells for adoptive immunotherapy of the poorly immunogenic BI6-BL6 (BL6) melanoma. C57BL/6 (B6) mice were immunized subcutaneously (s.c.) with 3 × 10⁶ viable BL6 tumor cells admixed with various doses of OK-432 ranging from 1 to 100 μg in the foot-pad. Draining popliteal lymph nodes (LNs) were harvested 7 days after immunization and LN cells were further sensitized with irradiated tumor cells in the presence of 60–300 IU/ml of IL-2 for 11 days. These in vitro sensitized (IVS) cells (2 × 10⁶) were transferred intravenously (i.v.) to B6 mice bearing 4-day pulmonary metastases established by i.v. injection of 2–4 × 10⁵ viable BL6 cells. The mice were also received intraperitoneally (i.p.) 4 × 10⁴ IU/day of IL-2 for 4 days after adoptive transfer. Transfer of IVS cells from mice immunized by s.c. injection of tumor cells admixed with 10 μg of OK-432 significantly reduced the numbers of BL6 pulmonary metastases compared with that of control IVS cells without the administration of OK-432 (P = 0.003). These effective IVS cells also significantly prolonged the survival of treated animals (P=0.003). Functional IVS cells required in vitro stimulation with tumor cells. However, addition of OK-432 in the vaccine resulted in no enhancement of in vitro cytotoxicity and no characteristic change of phenotype of IVS cells. These results suggest that in vivo priming of OK-432 facilitates the sensitization of tumor-reactive T-cells. The procedure of in vivo priming with OK-432 may be beneficial in the adoptive immunotherapy of melanoma.     

Hydrogen Peroxide-Releasing Function of Chemically Elicited and Immunologically Activated Macrophages: Differential Response to Wheat Germ Lectin and Concanavalin A

Various types of mouse peritoneal macrophages were studied for H₂O₂ release in the presence of wheat germ lectin or phorbol myristate acetate. Macrophages elicited 3 days before harvest by a single injection of thioglycolate, zymosan A, or a streptococcal preparation (OK-432) were highly responsive to wheat germ lectin, resulting in a marked increase in H₂O₂ release. However, immunologically activated macrophages induced by double injections of live and heat-killed BCG at 15 and 3 days before harvest or by double injections of zymosan A or OK-432 at 20 and 3 days before harvest did not show any significant response to wheat germ lectin. On the other hand, all macrophages tested responded well to phorbol myristate acetate by augmentation of H₂O₂ release. Concanavalin A inhibited wheat germ lectin- and phorbol myristate acetate-triggered H₂O₂ release from all types of macrophages, but inhibition was much more marked in the case of wheat germ lectin-stimulated H₂O₂ release. Succinylated concanavalin A (divalent concanavalin A) showed only slight suppressive action against macrophage H₂O₂ release, and prostaglandin E₁ and dibutyryl cyclic adenosine 3′, 5′-monophosphate caused depression of H₂O₂ release from OK-432-induced macrophages.     

Leukocyte hydrogen peroxide production in a surgical wound in mice. The effects of an amide local anaesthetic

The oxygen metabolism of polymorphonuclear leukocytes (PMN) is of importance in local tissue repair processes. Amide local anaesthetics are commonly used to relieve surgical wound pain. The cellular effects of local anaesthetics in vivo is poorly described in the literature. However, interactions between amide local anaesthetics and the oxygen metabolism of leukocytes have been reported. To extend that knowledge, this paper investigates the influence of lidocaine treatment on the production of hydrogen peroxide (H₂O₂) by leukocyte oxygen metabolism. A soft tissue chamber model in the mouse was used, allowing measurements of the H₂O₂ production spontaneously and after phorbol myristate acetate (PMA) addition, from two different leukocyte pools. Exudate leukocytes were generally more reactive to PMA stimulation in comparison to tissue chamber adherent leukocytes. Topically administered lidocaine significantly influenced the number of leukocytes in the wound exudate at 24 h postoperatively. Exudate leukocytes, topically exposed to lidocaine, showed an enhanced H₂O₂ production in comparison to leukocytes receiving lidocaine systemically. At 6 days, the viability and the H₂O₂ production differed significantly between the group receiving topically applied lidocaine in comparison to placebo. We conclude that the wound healing process may be effected by topically applied lidocaine, administered in clinical doses, at least via interference with leukocyte oxygen metabolism.