The role of mast cells in the infiltration phenomena in inflammation

On the model of the acute infectious peritonitis in rats it is shown that the previous osmotic depletion of the peritoneal mast cell population leads to the increased functional activity of neutrophils and decreased of monocytes of the inflammatory focus and blood. The results indicate that in the natural inflammatory conditions mast cells directly or indirectly inhibit neutrophils and stimulate monocytes, i.e. they are the modulators of leukocytic reaction.     

Recombinant human interleukin-11 does not affect functions of purified human neutrophils in vitro

Recombinant human interleukin-11 (rHu-IL-11) is a multifunctional cytokine with thrombopoietic activity and demonstrated clinical efficacy in treating chemotherapy-induced thrombocytopenia. rHu-IL-11 also exhibits anti-inflammatory activity and is currently in clinical trials for the treatment of several inflammatory diseases. As neutrophils are involved in both innate immunity and an acute inflammatory response, the effect of rHU-IL-11 on the function of human peripheral blood neutrophils in vitro was examined. rHu-IL-11 was not cytotoxic and did not induce superoxide anion production or the release of granular enzymes from resting neutrophils. Phagocytosis and chemotaxis were unaffected. rHu-IL-11 treatment did not block the response of neutrophils to stimulation. Pretreatment with rHu-IL-11 did not reduce production of IL-8 following activation with lipopolysaccharide (LPS) or zymosan A particles. Pretreatment with rHu-IL-11 did not affect the release of lysozyme and beta-glucuronidase in response to A23187 or PMA-stimulated production of superoxide anion. These results indicate that rHu-IL-11 does not directly modulate key functions of neutrophils in vitro.     

Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.     

Chemotactic migration triggers IL-8 generation in neutrophilic leukocytes

Neutrophils recovered from inflammatory exudates possess increased levels of IL-8, but exposure of neutrophils to chemoattractants results in only a modest stimulation of IL-8 generation. This study was undertaken to explore the hypothesis that IL-8 generation in these cells is dependent upon the process of migration. Neutrophils synthesized up to 30 times as much IL-8 during migration in response to a gradient of diverse chemoattractants than they did when stimulated directly by the attractants in the absence of a gradient. This IL-8 response was dependent on migration since it was not observed in cells exposed to concentration gradients of chemoattractants under conditions that prevented cell movement. While actinomycin-D (1 microg/ml) had little influence on the generation of IL-8 during chemotaxis, the protein synthesis inhibitor cycloheximide (10 microg/ml) markedly blunted the accumulation of cell-associated IL-8, suggesting that new protein synthesis from preexisting mRNA was responsible for the effect. Consistent with this interpretation, migrating cells incorporated over 10 times as much [3H]leucine into IL-8 as did nonmotile neutrophils exposed to chemoattractants. A substantial portion of the IL-8 generated during chemotaxis was released upon subsequent metabolic stimulation. Thus, the IL-8 synthesized during chemotaxis is uniquely positioned to exert a regulatory influence on inflammatory processes governed by neutrophilic leukocytes responding to inflammatory and infectious stimuli.     

The local role of tumor necrosis factor alpha in the modulation of neutrophil function at sites of inflammation

OBJECTIVE: To examine the hypothesis that tumor necrosis factor alpha (TNF-alpha) is an important local modulator of neutrophil function in the inflammatory microenvironment. DESIGN: In vitro studies of host defense. PATIENTS: A volunteer sample of healthy subjects. INTERVENTION: Exudative neutrophils were collected from skin-blister chambers and functionally compared with blood neutrophils. METHODS: Tumor necrosis factor alpha levels at sites of inflammation and neutrophil exudation were determined and compared with serum concentrations. Flow cytometry was used to evaluate neutrophil microbicidal activity and N-formyl-methionyl-leucyl-phenylalanine-induced changes in intracellular calcium and superoxide production. In vitro TNF-alpha was used to evaluate the nature and dose response of TNF-alpha-induced changes in neutrophil function. RESULTS: Exudative neutrophils have an increased responsiveness to subsequent N-formyl-methionyl-leucyl-phenylalanine stimulation, as determined by changes in intracellular calcium. Microbicidal activity and superoxide production are also up-regulated compared with circulating neutrophils. The exudative microenvironment contains TNF-alpha at local levels that are capable of significantly enhancing neutrophil host defense. CONCLUSIONS: Tumor necrosis factor alpha may serve to enhance neutrophil function at sites of inflammation. Neutrophils become more cytotoxic and have an enhanced ability to respond to weak environmental signals.     

The effect of dexamethasone on the reactions of the blood system in inflammation

On the model of carrageenan-induced acute aseptic peritonitis in rats it is shown that under influence of dexamethasone granulomonocytopoiesis, efflux of leukocytes to blood and their accumulation in inflammatory focus are increased and earlier completion of leukocytic reaction is observed and that the antiinflammatory effect of dexamethasone is mainly realized by the way of increasing of defence-adaptative blood system's reactions.     

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.     

The production of macrophage inflammatory protein-1 alpha by human basophils

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.     

The formation of blood system tolerance in rats to the repeated action of a stressor stimulus

Repeated immobilisation induced a biphasic dynamic of the blood system stress sensitivity: first, an increased neutrophilia, lymphocytopenia, bone marrow granulocyte depletion and activation of the erythropoiesis; second, a restitution of mature granulocyte-monocyte cells number in the bone marrow and accumulation of neutrophils in the spleen.     

Local vaginal therapies

It is known that polymorphonuclear leucocytes are deeply involved in the inflammatory complications of diabetes mellitus, showing many functional and biochemical abnormalities. Because adenine and guanine metabolites exert an important role in many metabolic aspects of phagocytic cells, we have investigated the pattern of purine metabolites during the respiratory burst of polymorphonuclear leucocytes in order to characterize any difference that may be significantly correlated with the abnormal neutrophil function of diabetic patients. The results obtained show clearly that polymorphonuclear leucocytes from diabetic patients are characterized by an abnormal pattern of purine nucleotides and their metabolites. In particular, the concentration of adenine and guanine triphosphates and the net amount of adenosine triphosphate hydrolysed during neutrophil stimulation by phorbol ester is higher in diabetic than in control cells. Moreover, higher values of adenosine monophosphate, inosine monophosphate and inosine have been found in diabetic cells. The behaviour of guanosine triphosphate is highly interesting. In fact, in addition to the higher concentration found in diabetic polymorphonuclear leucocytes, stimulation by phorbol ester induces a net decrease in guanosine triphosphate whereas control neutrophils show a slight increase. These findings have been associated with the ease with which diabetic neutrophils undergo metabolic activation and sustain an inflammatory response.     

Review: immunobiology of preeclampsia

Nitric oxide (NO) is a regulator of leukocyte adhesion in the microcirculation. This study was designed to examine the effects of a NO synthase inhibitor on neutrophil adhesion in the peritoneum, lung, liver, and kidney in a rat peritonitis model using a fluorescence microscopic method. Sprague-Dawley rats were given normal saline (control) or N omega-nitro-L-arginine methyl ester (L-NAME) at dosages of 10 mg/kg (N10) or 100 mg/kg (N100) (n = 66) intraperitoneally. One hour after pretreatment fluorescein-labeled neutrophils were infused without bacterial challenge (0 hr). Other rats received an injection of 10(7) Escherichia coli into the peritoneal cavity 1 hr after pretreatment. Labeled neutrophils were infused 1 and 5 hr after bacterial challenge. Just 2 min after neutrophil injection, blood samples were obtained and the animals were killed. Five peritoneal samples (omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Combined plasma nitrite/nitrate levels were determined. In another set of rats (n = 36), an arterial catheter was inserted after L-NAME treatment and bacterial challenge. At 0, 1, 5, and 12 hr after challenge, blood pressure, heart rate, and arterial blood gas data were measured. One hour after E. coli challenge, the number of neutrophils in the peritoneum was significantly lower in both L-NAME-treated groups than in the control group. In contrast, the number of labeled neutrophils in the lungs was significantly higher in the N100 group than in the control group. Neutrophil accumulation in the lungs and peritoneum at 0 and 5 hr and in the liver and kidney at 0, 1, and 5 hr did not differ among groups, nor did combined plasma nitrite/nitrate levels. L-NAME treatment had no influence on either hemodynamic or blood gas data. In conclusion, administration of L-NAME increases neutrophil adhesion in the lung, while decreasing that in the peritoneum. NO plays an important role in neutrophil adhesion at the inflammatory site, as well as in remote organs, during peritonitis. NO inhibition may be detrimental, due to neutrophil sequestration, in this peritonitis model.     

Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-alpha

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.     

The neuropeptide alpha-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro

The proopiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) has potent anti-inflammatory effects in all animal models of inflammation against which it has been tested. Understanding of the mechanism by which this occurs is incomplete, although there is recent evidence for alpha-MSH receptors in murine and human macrophages and for modulation of production of proinflammatory cytokines and related mediators by alpha-MSH. Because of the prominence of neutrophils in early stages of inflammatory reactions where alpha-MSH is effective, we examined human neutrophils for evidence of mRNA for alpha-MSH receptors and for inhibition of neutrophil chemotaxis. There was accumulation of mRNA for melanocortin receptor 1 (MC1) in RT/PCR product from neutrophils stimulated with interferon and LPS. In subsequent studies alpha-MSH inhibited migration of neutrophils from most normal volunteers when the cells were placed in FMLP or IL-8 gradients. The inhibition by alpha-MSH could be traced to alterations in cAMP in neutrophils. The presence of alpha-MSH receptor message in neutrophils is consistent with the established anti-inflammatory effects of the peptide. Direct inhibition of neutrophil chemotaxis likely contributes to the anti-inflammatory activity of alpha-MSH.     

Apoptosis of neutrophils and their elimination by Kupffer cells in rat liver

The fate of neutrophils in the peripheral circulation is poorly understood. In this study, the role of Kupffer cells in eliminating aged and apoptotic neutrophils was investigated. Liver, spleen, lung, and blood samples from Wistar rats were examined by light and electron microscopy, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method, and immunohistochemistry after the intravenous injection of OK-432, a streptococcal preparation. Neutrophils were trapped predominantly in the periportal and midzonal regions of hepatic lobules and were in contact with endothelial cells and Kupffer cells, or were surrounded by Kupffer cells. The trapping of neutrophils peaked after 6 hours. Apoptotic neutrophils, with or without buds, were found in the lumen of hepatic sinusoids as early as 6 hours, reached maximal levels after 12 hours, and represented greater than 60% of the total number of neutrophils in the liver. The presence of apoptotic neutrophils was correlated with the degree of neutrophil phagocytosis. Double-staining showed that TUNEL-positive neutrophils were phagocytosed or encircled by ED1- or ED2-positive Kupffer cells. In contrast, apoptosis and phagocytosis of neutrophils were rare in the spleen, lung, and peripheral blood. These results suggested that the appearance of apoptotic neutrophils in the hepatic sinusoids and their rapid clearance by Kupffer cells occurs after the invasion of bacteria (i.e., bacteremia or bacteriotoxemia) or the release of inflammatory mediators into the blood stream. These findings have important implications for the regulation of neutrophil homeostasis, the limitation of inflammation and tissue injury, and provide insight into the physiological removal of circulating, senescent neutrophils.     

Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils

BACKGROUND: As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. METHODS: Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. RESULTS: Aging was associated with a significant reduction in the Vmax for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the Km was noted in neutrophils from young but not from old volunteers. CONCLUSIONS: These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.     

Spontaneous listeric encephalitis in sheep. Electron microscopic studies

The brainstems of four sheep with spontaneous listeric encephalitis had scattered small foci of inflammatory cells (neutrophils or macrophages, or both) with scattered fragments of degenerating nerve fibers and glial cells. In extensive areas of malacia in the pons and medulla oblongata, there was loss of parenchyma with massive accumulation of macrophages, a few neutrophils, lymphocytes and plasma cells. In both types of lesions, phagocytes contained debris of myelin and axons, lipid vacuoles and occasionally bacteria. Neutrophils contained bacteria in phagocytic and digestive vacuoles. No bacteria were detected in macrophages but were detected in neurons and in one axon in tissue previously used for paraffin sections.     

Differential expression of two major cytokines produced by neutrophils, interleukin-8 and the interleukin-1 receptor antagonist, in neutrophils isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: Neutrophils have been shown to produce interleukin-8 (IL-8) and interleukin-1 receptor antagonist (IL-1ra) in large amounts compared with other cytokines. Since IL-8 has a proinflammatory action whereas IL-1ra is antiinflammatory, our objective was to examine the relative levels of production of these cytokines by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA). METHODS: We measured cytokine production using an enzyme-linked immunosorbent assay and analyzed messenger RNA accumulation in cells by Northern blot. RESULTS: SF neutrophils produced significantly more IL-8 and IL-1 beta, but significantly less IL-1ra, than peripheral blood neutrophils. CONCLUSION: These observations provide new information on the production of pro- and antiinflammatory molecules by neutrophils in the SF environment, and their possible role in RA.     

Body surface mapping during pacing at multiple sites in the human atrium: P-wave morphology of ectopic right atrial activation

A magnetic cell sorting system has been optimised for the purification of rainbow trout neutrophils using a monoclonal antibody (E3D9) raised against Atlantic salmon neutrophils. The purified neutrophils have good viability (85%) and purity (approximately 92%), and were functional in respiratory burst and migration assays. The isolated neutrophils responded rapidly to PMA stimulation, producing levels of superoxide anion (4.85 nmols superoxide min-1/10(6) cells) approximately twice as high as macrophages from the same species. In the migration assay, there was a four-fold increase in migrating cells using the purified neutrophils compared with unfractionated blood leucocytes, and a relatively high neutrophil migratory activity was seen in the absence of serum.     

Effect of theophylline on CD11b and L-selectin expression and density of eosinophils and neutrophils in vitro

The nonspecific phosphodiesterase inhibitor theophylline, widely used in asthma therapy, may cause a decrease in inflammatory responses of airways. In asthma, eosinophils migrate to the airway wall and become activated. Activated eosinophils are characterized by low cell density, as well as increased expression of CD11b and reduced expression of L-selectin, two adhesion molecules involved in transendothelial migration. To study the anti-inflammatory effect of theophylline on granulocyte adhesion molecules in vitro, the platelet-activating factor (PAF)-induced density shift was determined by density centrifugation and the modulation of CD11b and L-selectin expression by flow cytometry on eosinophils and neutrophils in human whole blood. A relatively high concentration of theophylline (10(-3) M) inhibited the increase in the percentage of hypodense eosinophils and neutrophils in whole-blood samples after PAF stimulation in vitro. A more pharmacological concentration (10(-4) M) inhibited the CD11b upregulation and L-selectin shedding induced by PAF (10(-7) M) on both eosinophils and neutrophils. The effect of isoproterenol on the inhibitory effect of theophylline was mainly additive, but a small synergistic effect could not be excluded. In conclusion theophylline can attenuate eosinophil and neutrophil activation in vitro at the level of adhesion molecule expression and changes in cell density. This may have implications for transendothelial migration of these cells in asthma.