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PubMed1998

Methylprednisolone-induced neutrophil leukocytosis--down-modulation of neutrophil L-selectin and Mac-1 expression and induction of granulocyte-colony stimulating factor.

The mechanisms underlying corticosteroid-induced neutrophil leukocytosis are not fully understood; however, leukocyte/endothelial cell adhesion molecule interactions are known to be key to the movement of neutrophils within and out of the vasculature. This study was designed to investigate the effects of corticosteroids on neutrophil adhesion molecules in relation to neutrophil leukocytosis. Circulating neutrophil counts, neutrophil L-selectin and Mac-1 expression (measured by flow cytometry), soluble L-selectin, and granulocyte-colony stimulating factor concentrations were determined in 15 multiple sclerosis patients receiving intravenous methylprednisolone prior to and at 6 and 24 h following the initial 500-mg dose. A follow-up sample was obtained 48 h after the 5-day therapeutic course. Neutrophil counts were elevated at 6 h (threefold) and 24 h (twofold). This was associated with a 40% reduction in L-selectin expression at 6 and 24 h and a 35% reduction in Mac-1 expression at 6 h. Serum granulocyte-colony stimulating factor levels were increased (6 h: threefold; 24 h: twofold), whereas soluble L-selectin concentrations were unaltered. All of the above parameters had returned to basal levels in the follow-up sample. Short-term in vitro cultures (6 and 24 h) of blood samples from untreated multiple sclerosis patients and controls with 0.01 mg/ml methylprednisolone resulted in minimal reductions in neutrophil L-selectin and Mac-1 and no change in soluble L-selectin. Granulocyte-colony stimulating factor induced Mac-1 expression in a dose-dependent manner, whereas L-selectin expression was unaffected or reduced at high concentrations. Reduction in neutrophil L-selectin and Mac-1 expression following methylprednisolone infusion may cause decreased adhesion of marginated neutrophils and/or reduced capacity of neutrophils to migrate from the vasculature. Additionally, the induction of granulocyte-colony stimulating factor may contribute to neutrophil production and release into the circulation.

Down-Regulation↗

PubMed2005

Gene expression signatures in neutrophils exposed to glucocorticoids: a new paradigm to help explain "neutrophil dysfunction" in parturient dairy cows.

Neutrophils are the first line of immunity against most pathogens that infect cattle. These normally short-lived white blood cells develop from myeloid-lineage cells in bone marrow. Upon maturation, bone marrow neutrophils are released into the circulation where they marginate on inflamed blood vessel endothelial cells and migrate through them into the area of infection. Once migrated, neutrophils do not reenter the circulation, but rather, perform their bactericidal functions and die by apoptosis in the tissue. The cytokine and hormonal milieu of the blood and extracellular tissue fluid can influence neutrophil development and immunity-related activities, but the molecular basis of these phenotypic changes and physiological benefits or drawbacks of them are poorly understood. In the current paper, we review new gene expression information that resulted from two of our functional genomics studies designed to evaluate effects of glucocorticoid hormones on bovine neutrophils. This work provides one model to describe complex changes that occur in neutrophils as the cells respond to glucocorticoids, which might act to alter the cells' functional priorities and tip the delicate balance between health and disease during stress, including at parturition. A bovine immunobiology microarray and real time RT-PCR were used to study blood neutrophils collected during the natural surge of endogenous glucocorticoid (cortisol) in parturient dairy cows and bone marrow neutrophils collected from glucocorticoid (dexamethasone)-treated dairy steers. The gene expression signatures we observed led us to perform additional phenotyping of the neutrophils and correlation analyses, which together painted a picture suggesting that glucocorticoids have key roles in modulating neutrophil development, life span, and tissue defense functions during parturition and hormone therapy. Based on these observations, we postulate that glucocorticoids orchestrate adaptive changes in the entire neutrophil system that support increased cell numbers and longevity in blood and heightened remodeling activity in tissues, while at the same time decreasing some important antimicrobial defense activities of the cells. Thus, our functional genomics studies have enabled us to elucidate multiple consequences of neutrophil exposure to glucocorticoids, highlighting a probable role for this interaction in the induction of parturition and partly explaining why some parturient dairy cows may experience heightened incidence and severity of inflammatory diseases like mastitis.

Animals↗

PubMed2002

Protein kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neutrophils and changes during myeloid maturation in chronic myeloid leukemia.

Protein kinase C (PKC) is reported to play a role in maturation of the myeloid cell and functions of the mature neutrophil. The neutrophils in chronic myeloid leukemia (CML) exhibit defects in several functions. As a step towards understanding the role of PKC in the defects in function of the leukemic cells, this study investigates the expression of PKC isoforms, their subcellular distribution, levels and kinase activity in the normal and leukemic neutrophils. It also investigates changes in representative PKC isoforms during myeloid maturation. This study confirms the presence of PKC alpha, beta and delta and shows, for the first time, the presence of non conventional PKC isoform theta, atypical PKC isoform lambda/iota and PKC isoform mu in normal human neutrophils. In unstimulated cells all the detected PKC isoforms showed a predominantly cytosolic localisation in normal and CML neutrophils. Cytosol-membrane distribution of PKC alpha and delta were significantly altered in leukemic neutrophils as compared to normal cells. Cytosolic levels of all PKC isoforms were reduced in CML neutrophils with PKC alpha, beta, iota, theta, and mu showing a significant decrease. Cytosolic levels of PKC delta contrary to the trend observed for other PKC isoforms showed a slight increase in CML cells, while its membrane levels were significantly reduced in CML neutrophils. Total PKC kinase activity in CML neutrophil cytosol was significantly reduced, while specific kinase activity of two representative isoforms, PKC alpha and delta, from normal and CML neutrophils were similar, thereby increasing the significance of the altered levels of PKC isoforms in CML, and highlighting their role in the defects in function exhibited by the leukemic neutrophils. The levels of PKC delta and iota increased and decreased respectively as the leukemic myeloid cell matured from the blast to the neutrophil, while the levels of PKC alpha and beta were not altered. This suggests a role for PKC delta and iota in the maturation of the leukemic myeloid cell.

Cell Fractionation↗

PubMed1992

Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils.

To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common beta chain (CD18), or the distinct alpha chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene. Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H2O2; H2O2 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, beta 2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKM1, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H2O2 or O2- release from neutrophils. However, mAbs against LFA-1 or gp150/95 triggered both H2O2 and O2- release from neutrophils. Stimulation of neutrophils respiratory burst by both anti-CD18, and anti-LFA-1 or gp150/95 mAbs was totally inhibited by the microfilaments disrupting agent, cytochalasin B, and by a permeable cAMP analogue. While the capability to activate neutrophil respiratory burst was restricted to anti-LFA-1 and gp150/95 mAbs, we observed that mAbs against all members of leukocyte integrins, including CR3, were able to trigger neutrophil spreading. These findings indicate that, in neutrophils, all three leukocyte integrins can generate signals activating spreading, but only LFA-1 and gp150/95 can generate signals involved in activation of the respiratory burst. This observation can be relevant to understand the mechanisms responsible for the activation of neutrophil respiratory burst by tumor necrosis factor-alpha, which has been shown to be strictly dependent on expression of leukocyte integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. Wright. 1989. J. Cell Biol. 109:13411349.

Antibodies, Monoclonal↗

PubMed1979

Neutrophil function in anergic surgical patients: neutrophil adherence and chemotaxis.

Skin test anergy (A) to recall antigens identifies surgical patients at high risk for sepsis. We studied neutrophil function in such patients to assess any alteration in their host defense mechanisms. Neutrophil adherence was measured with a modified adherence assay capable of measuring the adherence of neutrophils in whole blood or purified neutrophil suspensions, and neutrophil chemotaxis was assessed by the Boyeden technique. Twenty-one laboratory controls had a neutrophil adherence of 71.5 +/- 3.8% (mean +/-SD) and chemotaxis of 128.1 +/- 2.4,micro (mean +/-SD). Fifty-four hospitalized patients with normal skin tests had neutrophil adherence of 72.5 +/- 13.1% (p ~ 0.5 relative to control) and chemotaxis of 123.3 +/- 3.1 micro (p ~ 0.5). Twenty three relatively anergic patients had values of 84.3 +/- 7.9% (p < 0.001) and 103.7 +/- 2.0 micro (p < 0.001). Forty five A patients had adherence of 85.0 +/- 7.0% (p < 0.001) and chemotaxis of 90.4 +/- 2.9 micro (p < 0.001). The correlation coefficient between increased neutrophil adherence and decreased chemotaxis r = 0.81 has p < 0.0005. A factor which increased the adherence of normal control neutrophils was found in the plasma but not the serum of anergic patients. Inhibitors of control neutrophil chemotaxis have been shown in both serum and plasma of patients with decreased autologous neutrophil chemotaxis. We propose that this altered neutrophil function (possibly with other defects) in anergic patients may compromise their host defenses and render them susceptible to infection.

Adolescent↗

PubMed2000

Tilmicosin induces apoptosis in bovine peripheral neutrophils in the presence or in the absence of Pasteurella haemolytica and promotes neutrophil phagocytosis by macrophages.

Pathogen virulence factors and inflammation are responsible for tissue injury associated with respiratory failure in bacterial pneumonia, as seen in the bovine lung infected with Pasteurella haemolytica. Tilmicosin is a macrolide antibiotic used for the treatment of bovine bacterial pneumonia. Recent evidence suggests that tilmicosin-induced neutrophil apoptosis may have anti-inflammatory effects. Using bovine leukocytes, we sought to define whether live P. haemolytica affected tilmicosin-induced neutrophil apoptosis, assessed the proapoptotic effects of tilmicosin in comparison with other drugs, and characterized its impact on phagocytic uptake of neutrophils by macrophages. Induction of apoptosis in the presence or absence of P. haemolytica was assessed by using an enzyme-linked immunosorbent assay for apoptotic nucleosomes. In addition, fluorescent annexin-V staining identified externalized phosphatidylserine in neutrophils treated with tilmicosin, penicillin, ceftiofur, oxytetracycline, or dexamethasone. Neutrophil membrane integrity was assessed by using propidium iodide and trypan blue exclusion. As phagocytic clearance of apoptotic neutrophils by macrophages contributes to the resolution of inflammation, phagocytosis of tilmicosin-treated neutrophils by esterase-positive cultured bovine macrophages was assessed with light microscopy and transmission electron microscopy. Unlike bovine neutrophils treated with penicillin, ceftiofur, oxytetracycline, or dexamethasone, neutrophils exposed to tilmicosin became apoptotic, regardless of the presence or absence of P. haemolytica. Tilmicosin-treated apoptotic neutrophils were phagocytosed at a significantly greater rate by bovine macrophages than were control neutrophils. In conclusion, tilmicosin-induced neutrophil apoptosis occurs regardless of the presence or absence of live P. haemolytica, exhibits at least some degree of drug specificity, and promotes phagocytic clearance of the dying inflammatory cells.

Animals↗

PubMed2001

Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity.

BACKGROUND: Increased numbers of apoptotic neutrophils, and impaired monocyte/macrophage clearance of apoptotic cells, have been demonstrated in systemic lupus erythematosus (SLE). CD44 is implicated in the clearance of apoptotic neutrophils. OBJECTIVE: To determine the expression of CD44 on peripheral blood monocytes and neutrophils in SLE, and examine the relations with disease activity and numbers of circulating apoptotic neutrophils. METHODS: Peripheral blood was sampled from 31 patients with SLE, 19 healthy normal subjects, and 19 patients with rheumatoid arthritis (RA). Monocyte and neutrophil density of surface CD44 expression was determined by immunofluorescence labelling and flow cytometry, and results expressed as mean channel fluorescence (MCF) values. Neutrophil apoptosis was measured by morphology in 15 patients with SLE, nine with RA, and six normal subjects. RESULTS: Monocyte CD44 expression was significantly lower in SLE (median MCF 4.71) than in healthy normal subjects (median MCF 5.61) and controls with RA (median MCF 5.39). Neutrophil CD44 expression was also significantly lower in SLE (median MCF 1.95) than in healthy normal subjects (median MCF 2.37) and controls with RA (median MCF 2.60). Monocyte, but not neutrophil, CD44 expression correlated negatively with the percentage of apoptotic neutrophils. There was no significant correlation of monocyte or neutrophil CD44 expression in SLE with disease activity or damage. CONCLUSIONS: Monocyte and neutrophil CD44 expression is reduced in SLE, and this may contribute to the impaired recognition and clearance of apoptotic neutrophils by monocyte derived macrophages.

Apoptosis↗

PubMed2000

Neutrophils mediate acute lung injury in rabbits: role of neutrophil elastase.

We investigated the roles of neutrophil and neutrophil elastase in acute lung injury (ALI) to elucidate the mechanism of ALI. We designed two protocols. Protocol I: Experimental ALI was induced by endotoxin (0.02 mg/kg) and platelet-activating factor (8 microg/kg/4 h) in untreated rabbits (control group I), in neutropenic rabbits pretreated with nitrogen-N-oxide hydrochloride, and in untreated rabbits infused with a neutrophil elastase inhibitor (ONO-5046; 20 mg/kg/4 h). Protocol II: ALI was induced by smaller doses of endotoxin (0.015 mg/kg) and platelet-activating factor (7 microg/kg/4 h) than those used in protocol I in untreated rabbits (control group II), in neutrophilic rabbits pretreated with human recombinant granulocyte colony-stimulating factor, and in neutrophilic rabbits infused with ONO-5046 (as in protocol I). The severity of ALI was assessed by the protein concentration, the elastase activity in the bronchoalveolar lavage fluid, and the histologic pulmonary edema ratio. The degree of pulmonary neutrophil accumulation was assessed by pulmonary myeloperoxidase activity and histological findings. Both ALI and pulmonary neutrophil accumulation were suppressed by neutropenia (protocol I), while they were exacerbated by neutrophilia (protocol II). The neutrophil elastase inhibitor could suppress ALI, but it could not suppress pulmonary neutrophil accumulation in both untreated and neutrophilic rabbits (protocols I and II). These findings indicate that neutrophils play an important role in the pathogenesis of ALI via neutrophil elastase.

Acute Disease↗

PubMed1987

The pulmonary vascular sequestration of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit.

Endotoxemia causes neutrophil sequestration in the pulmonary vascular bed. Such sequestration may be a critical initiating event in the generation of microvascular injury, although the mechanisms that lead to this localization are not understood. To investigate these phenomena, the following study employed intravenous pulses of 111Indium-tropolonate-labeled neutrophils (111In-neutrophils), which circulated in the rabbit with normal kinetics and responded in a manner indistinguishable from unlabeled, circulating neutrophils in response to an intravenous injection of purified endotoxic lipopolysaccharide (LPS) or epinephrine. Pulmonary sequestration of 111In-neutrophils was assessed by quantitative external gamma camera scintigraphy of a lung suprahilar region of interest. Noninvasive assessment of radioactivity by this method accurately reflected total lung radioactivity, which was shown by autoradiography to be confined to the injected 111In-neutrophils. Intravenously administered LPS caused a marked, dose-dependent sequestration of 111In-neutrophils in the pulmonary vasculature, and exhaustive ultrastructural autoradiography showed discretely radiolabeled neutrophils located within pulmonary capillaries. A distinct effect was seen with an intravenous injection of as little as 100 ng per rabbit (i.e., 500 pg/ml blood). A 5-min ex vivo pretreatment of 111In-neutrophils with 10 ng to 10 micrograms/ml LPS in heat-inactivated plasma (which resulted in retention of as little as 500 pg LPS per 10(7) neutrophils) also caused dose-dependent pulmonary sequestration of the pretreated 111In-neutrophils but did not cause generalized neutropenia in recipient rabbits. There was no evidence of complement activation on the surface of pretreated neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

PGE1 inhibits neutrophil adherence and neutrophil-mediated injury to cultured endothelial cells.

Prostaglandin E1 (PGE1) has been shown to inhibit acute inflammatory reactions involving vascular permeability changes and subsequent tissue damage caused by immune complex deposition and other inflammatory mediators. These effects have been postulated to be due to functional changes induced in circulating neutrophils by PGE1. We evaluated the ability of PGE1 (1 to 100 microM) to protect endothelial cells (EC) from neutrophil injury induced by C5a or phorbol myristate acetate (PMA). The injury to endothelial monolayers was quantitated by 51Cr loss after an 18-h incubation. In the presence of PGE1, there was a concentration-dependent inhibition of C5a- or PMA-stimulated human neutrophil injury to EC. The protective effect was more effective by pretreatment of neutrophils than EC. Since neutrophil adherence to endothelial cells is thought to be an early event in the sequence resulting in injury to vascular endothelium, we next evaluated the effect of PGE1 on neutrophil adherence to plastic surfaces and EC. In a similar fashion, there was a concentration-dependent inhibition of neutrophil adherence to plastic and EC following stimulation with PMA (10 ng/ml), human C5a (5 micrograms/ml) or formyl-met-leu-phe (FMLP, 2 x 10(-7) M). To investigate a possible mechanism of inhibition of neutrophil adherence to plastic surfaces or EC, expression of the neutrophil surface protein Mo1, previously shown to be associated with increased granulocyte adherence, was measured by fluorescence flow cytometry. Although basal levels of Mo1 expression by unstimulated neutrophils were diminished by PGE1 treatment, there was no significant inhibition of PMA- or FMLP-stimulated Mo1 expression in PGE1-treated neutrophils compared with neutrophils stimulated in the absence of PGE1.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprostadil↗

PubMed1992

Neutrophil function as related to neutrophil-endothelial cell interactions.

Advances have recently been made in several areas related to neutrophil-mediated tissue damage. Certain chemotactic receptors have been cloned, their relationship to G proteins examined, and their mechanisms of downregulation partly elucidated. Downregulation of chemotactic receptors could be useful in controlling the damage inflicted by neutrophils on nearby tissues. Studies of neutrophil adhesion to endothelium have revealed two classes of adhesion proteins: selectins, and the integrin/integrin receptor system. Selectins are involved in neutrophil margination, while the integrin system participates in the egress of neutrophils into regions of inflammation. The microfilaments are strands of polymerized actin which form a network that pervades the neutrophil cytoplasm. On activation of the neutrophil, changes occur in the state of the microfilament network that appear to act in combination with the integrin/integrin receptor system to cause neutrophils to be retained in capillaries of tissues reperfused after hypoxia. To the extent that reperfusion injury is caused by these retained neutrophils, prevention of their effect may require measures directed at the microfilament network as well as the integrin/integrin receptor system. With regard to mechanisms of neutrophil-mediated tissue damage, recent studies have emphasized the extent to which oxidative systems interact with proteases to augment the damage inflicted by activated neutrophils. The unique efficacy of secretory leukoprotease in preventing neutrophil-mediated proteolysis is discussed.

Actin Cytoskeleton↗

PubMed1992

Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo. Neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells.

We have previously demonstrated that the murine colon adenocarcinoma C-26 cell line transduced with the human gene for the granulocyte CSF (G-CSF) loses tumorigenic activity through a mechanism that involved massive targeting of neutrophils at the site of tumor injection. The suppression of tumorigenicity by G-CSF was limited to the G-CSF-producing cells and was not transferred to nonproducing C-26 cells in a mixed tumor transplantation assay. We present direct evidence that neutrophils are involved in this phenomenon. We firstly examined, by electron microscopy (EM), the morphology of tumor infiltrates obtained 2, 5, and 10 days after s.c. injection of a mixture of G-CSF-producing and -nonproducing C-26 cells into syngeneic BALB/c mice. The EM analysis showed at 5, but not at 2 or 10 days, the presence of neutrophils in intimate contact with tumor cells. We then investigated whether neutrophils discriminate between G-CSF-producing and -nonproducing C-26 cells. To this aim, C-26 cells were transduced, via retroviral vector, with the Escherichia coli LacZ gene and mixed tumor transplantation assays were performed by injecting a mixture of G-CSF-producing beta-gal- and G-CSF-nonproducing beta-gal+ C-26 cells at different ratios. Histologic and EM analysis of the tumors growing at the site of injection were carried out. Five days after injection, treatment with x-gal revealed, at the histochemical level, the presence of neutrophils around G-CSF producing beta-gal- cells; cell-cell contacts and fusion of cell membranes were detected by EM only between neutrophils and G-CSF-producing cells. In vitro experiments, performed in Boyden chambers, confirmed that the G-CSF produced by C-26 cells was a chemoattractant for neutrophils. In addition, a colorimetric, cytostatic assay revealed that neutrophils were able to inhibit the growth of G-CSF-producing but not of G-CSF-nonproducing C-26 cells. Thus the tumor take after injection of G-CSF-producing C-26 cells seems to be controlled in situ through two major mechanisms namely neutrophil chemotaxis and neutrophil-mediated tumor inhibition. The results indicate that neutrophils can discriminate between G-CSF-producing and -nonproducing tumor cells and that neutrophils infiltrate the tumor mixture as long as G-CSF-producing cells are present.

Adenocarcinoma↗

PubMed1995

Expression of neutrophil activation markers and neutrophil adhesion to chondrocytes in rheumatoid arthritis patients: relationship with disease activity.

Neutrophil infiltration in synovial fluid is an important step in inflammation characterizing rheumatoid arthritis (RA). In this study, the activation and functional state of neutrophils in the blood and synovial fluid of patients with rheumatoid arthritis were compared: mean density of neutrophil activation markers CD11b, CD18 and L-selectin was measured with a flow cytometer, and adhesion to chondrocytes using a fluorimetric assay. No significant differences between control and patient peripheral blood neutrophils were observed. When comparing neutrophils of patient peripheral blood with paired synovial fluid, an increase in CD11b (p = 0.008) and a decrease in L-selectin (p = 0.008) were measured. For neutrophils of control and patient peripheral blood, fMet-Leu-Phe stimulation induced upregulation of CD11b (resp p = 0.007 and p = 0.008) and CD18 (resp p = 0.005 and p = 0.01). In the synovial fluid, no significant increase in CD11b and CD18 could be induced with fMet-Leu-Phe. Percentages of adherent neutrophils were comparable between controls and patients, both in peripheral blood and synovial fluid. Adhesion to chondrocytes of peripheral blood neutrophils of patients was correlated with clinical (Ritchie) and biological (erythrocyte sedimentation rate) parameters (resp r = 0.67, r = 0.73). In conclusion, these results demonstrate that peripheral blood neutrophil adhesion to chondrocytes was correlated with active disease, and that synovial fluid neutrophils were activated in vivo. These findings provide further evidence for the contributing role of neutrophils in articular destruction in RA.

Adult↗

PubMed2002

Autocrine production of epithelial cell-derived neutrophil attractant-78 induced by granulocyte colony-stimulating factor in neutrophils.

Whereas mobilization to inflammatory sites is an important function of neutrophils, it remains to be determined whether granulocyte colony-stimulating factor (G-CSF) stimulates the mobilization of neutrophils to the inflammatory sites. This study compared the expression of more than 9000 genes in neutrophils treated with and without G-CSF with the use of a DNA microarray system to determine the effects of G-CSF on the function of neutrophils. It was found that messenger RNA expression of epithelial cell-derived neutrophil attractant-78 (ENA-78), which has been reported to be a chemotactic factor for neutrophils, was induced by G-CSF in neutrophils. The study demonstrated that the supernatant of G-CSF-treated neutrophils induced the chemotaxis of neutrophils and that anti-ENA-78 antibody and anti-CXCR-2 antibody inhibited the chemotaxis. These data suggest that G-CSF may enhance the mobilization of neutrophils and consequently augment the accumulation of neutrophils in the inflammatory sites through the secretion of ENA-78.

Autocrine Communication↗

PubMed2004

The neutrophil, not the tumor: serum CA 15-3 elevation as a result of granulocyte--colony-stimulating factor-induced neutrophil MU1C overexpression and neutrophilia in patients with breast carcinoma receiving adjuvant chemotherapy.

BACKGROUND: Patients with resected breast carcinoma who received granulocyte-colony-stimulating factor (G-CSF)-supported adjuvant chemotherapy exhibited an increase in their serum CA 15-3 levels. The authors investigated the role of G-CSF-induced neutrophil MUC1 expression in this setting. METHODS: Twenty-two patients with resected early breast carcinoma and 6 patients with high-grade lymphoma received chemotherapy cycles with or without G-CSF support. When given, G-CSF was administered for either 5 or 10 days per cycle. Immunocytochemical staining and flow cytometric analysis of peripheral blood neutrophils and bone marrow myeloid cells for MUC1 epitopes were performed during treatment. RESULTS: At baseline, the median serum CA 15-3 was 16 U/mL, with weak MUC1 expression in peripheral neutrophils (median immunocytochemical score [ICCS] = 40, flow cytometric score [FCS] = 211 antibody molecules per neutrophil). For patients receiving chemotherapy cycles with 5-day G-CSF support, median CA 15-3 levels increased moderately (median = 28 U/mL; P = 0.016) and absolute neutrophil counts (ANC) did not increase, whereas ICC staining showed a moderate increase (median ICCS = 105; P = 0.015). For patients receiving chemotherapy cycles with 10-day G-CSF, serum CA 15-3 levels increased 2-4-fold from baseline levels and reached abnormal levels (median = 47; P < 0.0005) and the ANC increased (median = 21,400/mm(3); P = 0.007), whereas significant induction of MUC1 expression occurred in the cell membrane and mostly in the cytoplasm of neutrophils (median ICCS = 162; P = 0.001). Flow cytometry detected increased cytoplasmic, but not surface, neutrophil MUC1 expression in the 10-day G-CSF group only (baseline median FCS = 3975, 4th cycle median = 6327 molecules per cell; P = 0.028). In the bone marrow, induction of MUC1 expression was observed in the 10-day G-CSF group only in band forms and neutrophils, but not in more immature myeloid cells. Serum CA 15-3 levels and ICC score were found to demonstrate a linear relation. When ICCS and ANC were incorporated in a combined score, its relation with serum CA 15-3 levels demonstrated a parabolic (cubic) pattern. Serum CA 15-3 levels, ANC, and neutrophil MUC1 staining returned to baseline after the completion of therapy. No excess of malignant recurrences were observed. CONCLUSIONS: Women with resected breast carcinoma who received G-CSF-primed chemotherapy showed serum CA 15-3 elevation due to an increase in peripheral blood neutrophil number and induced neutrophil cytoplasmic MUC1 expression, which was caused by G-CSF. Physicians should be aware of this interaction.

Adenocarcinoma↗

PubMed1991

Neutrophil erythrotoxicity induced by phorbol myristate acetate: mechanisms involved in neutrophil activation.

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2-0.5 ng/ml) for 18 hr at 37 degrees C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.

Antigen-Antibody Complex↗

PubMed1989

Enhancement of normal neutrophil chemiluminescence by chronic granulomatous disease neutrophils.

Neutrophils and other phagocytic cells from patients with chronic granulomatous disease (CGD) lack the ability to generate reactive oxygen intermediates (ROI), although other phagocytic functions appear to be intact. The effects of CGD neutrophils on the ability of normal neutrophils to produce ROI as measured by luminol-enhanced chemiluminescence (CL) were examined. Normal neutrophils (2 x 10(5)) had a peak CL response to phorbol myristate acetate (PMA; 20 ng/ml) of 6.5 +/- 0.9 mV, while the CL response to CGD neutrophils was completely absent. However, the addition of CGD neutrophils (8 x 10(5)) to normal neutrophils (2 x 10(5)) markedly increased the peak CL response to PMA to 11.0 +/- 1.1 mV (P less than 0.001). The peak response of normal neutrophils (2 x 10(5)) alone to the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-6) M) was 9.0 +/- 1.1 mV, and this increased to 22.2 +/- 3.5 mV (P less than 0.001) when 8 x 10(5) CGD neutrophils were added and to 18.9 +/- 3.6 mV (P less than 0.005) when 4 x 10(5) CGD neutrophils were added. Thus, CGD neutrophils increase the release of ROI from normal cells, suggesting nonoxidative regulatory factors in ROI production.

Acridines↗

PubMed2006

C-reactive protein products generated by neutrophil elastase promote neutrophil apoptosis.

BACKGROUND: C-reactive protein (CRP), a prototypical acute phase protein, is increased as much as 1000-fold during acute inflammation, suggesting its biological role in that process. CRP can be modified at sites of inflammation where proteases have been released by neutrophils migrating to tissues. In this study, we investigated whether native CRP and neutrophil elastase-digested products of CRP modulate the rate of neutrophil apoptosis. METHODS: Neutrophils were isolated from venous blood of healthy volunteers and incubated with either native CRP (1, 10, or 30 microg/mL) or CRP digested with neutrophil elastase (1 U/mL). After 6 and 24 h of incubation, neutrophils were harvested and examined for apoptosis under light microscopy. RESULTS: We found that CRP degradation products generated by neutrophil elastase, but not native CRP, promoted neutrophil apoptosis and cell death. The rate of apoptosis was not affected by the addition of elastase that was not incubated with CRP. CONCLUSIONS: These results suggest that CRP degradation products generated by neutrophil elastase promote neutrophil apoptosis. Cleavage of CRP by neutrophil elastase may offer protection from inflammatory injury.

Apoptosis↗