Analysis of the functional characteristics of L-selectin and its expression on normal and CD18-deficient bovine neutrophils

In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18-deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly increased (P < 0.05) in four normal calves at 5-20 min after in vivo administration of epinephrine; however, no significant increase of neutrophils was found in three calves with bovine leucocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from three calves with BLAD was 61-77% of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate caused a marked decrease in the expression of L-selectin on neutrophils from both normal and BLAD calves. The sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased. Following stimulation with aggregated IgG, the transient phase of [Ca2+]i in neutrophils from normal and BLAD calves was increased; however, the sustained phase of [Ca2+]i in BLAD neutrophils was significantly lower (P < 0.05) than that of controls. Sulfatide-induced O2- production and chemiluminescent response in neutrophils from calves with BLAD were 48-51% of those of normal calves and were inhibited by genistein and wortmannin, respectively, in a dose-dependent manner. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from BLAD calves stimulated with sulfatides was 57% of that of controls. The degree of L-selectin expression on neutrophils was correlated with the intracellular signalling events and the related superoxide production.     

Possible exposure of leucine aminopeptidase on the cell surface of rabbit blood neutrophils by digitonin treatment

The sensitivity of leucine aminopeptidase to diazotized sulfanilic acid (DSA) was compared between neutrophils from blood and peritoneal exudates of rabbit. The leucine aminopeptidase activity of peritoneal neutrophils was inhibited about 40% by DSA, whereas that of blood neutrophils was not inhibited at all by the reagent. However, pretreatment of blood neutrophils with digitonin in the presence and in the absence of divalent cations rendered leucine aminopeptidase sensitive to DSA to the same extent as peritoneal neutrophils, without affecting the cell viability and lactate dehydrogenase activity. These findings seem to indicate that the leucine aminopeptidase of blood neutrophils, which is normally inaccessible to DSA, was exposed on the cell surface by digitonin treatment.     

Generation of biologically active C-reactive protein peptides by a neutral protease on the membrane of phorbol myristate acetate-stimulated neutrophils

The association of human C-reactive protein (CRP) with nonstimulated and PMA-stimulated human neutrophils and the concomitant degradation of CRP (monitored by TCA-soluble peptides and SDS-PAGE analysis) has been studied. Maximum association of 125I-labeled CRP with neutrophils and 125I-labeled CRP degradation during association with these cells was achieved by stimulating the neutrophils with PMA at 10 ng/ml; a concentration in which azurophil granule release was not significant. For PMA-stimulated neutrophils, the association of 125I-labeled CRP was 1.8 times higher and PMA-stimulated neutrophil-mediated degradation of the ligand was three times faster than that for nonstimulated cells. The neutrophil-associated 125I-labeled CRP in the absence and presence of PMA proved on SDS-PAGE analysis to be approximately 50% degraded. There was a positive correlation between the extent of CRP degradation and the association of 125I-labeled CRP with neutrophils. In addition to generation of neutrophil associated CRP intermediates, small soluble CRP peptides were generated during association of CRP with neutrophils. These peptides inhibited superoxide production from opsonized zymosan-activated neutrophils by approximately 40% at 10 micrograms/ml. 125I-labeled CRP degradation mediated by nonstimulated neutrophils, and neutrophil-conditioned medium (from both non-stimulated and PMA-stimulated cells) was inhibitable by alpha 1-antitrypsin and approximately seven times less at 1 h than that occurring during 125I-labeled CRP-association with PMA-stimulated neutrophils. The degradation of 125I-labeled CRP mediated by PMA-stimulated neutrophils was not fully inhibitable by alpha 1-antitrypsin. The data point to the involvement of a membrane-associated serine protease, which is maximally activated by PMA, in the degradation of 125I-labeled CRP during association with neutrophils. Our results indicate that at an inflammatory site CRP-derived peptides can be produced that inhibit the action of activated neutrophils.     

Human neutrophil cytosolic activation factor of the NADPH oxidase. Characterization of activation kinetics

The kinetics of sodium dodecyl sulfate-induced activation of respiratory burst oxidase (NADPH oxidase) in a fully soluble cell-free system from resting (control) or phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system containing solubilized membranes and cytosol fractions (cytosol) derived from control neutrophils (control cell-free system), the values of Km and Vmax for NADPH of the NADPH oxidase from control neutrophils continuously increased with increasing concentrations of cytosol, but with increasing concentrations of solubilized membranes from the control neutrophils, Km values continuously decreased, suggesting cytosolic activation factor-dependent continuous changes in the affinity of NADPH oxidase to NADPH. In a cell-free system containing solubilized membranes and cytosol prepared from PMA-stimulated neutrophils, NADPH oxidase was not activated after the addition of NADPH. However, cytosol from control neutrophils activated the NADPH oxidase of PMA-stimulated neutrophils in a cell-free system. Cytosol from PMA-stimulated neutrophils did not activate the control neutrophil oxidase, although it contained no inhibitors of NADPH oxidase activation. The results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted with an increasing period of time after the stimulation of neutrophils, and that the affinity of PMA-stimulated neutrophil NADPH oxidase to NADPH may almost be the same as that of control neutrophil oxidase. It was concluded that the affinity of NADPH oxidase to NADPH was closely associated with interaction between solubilized membranes and cytosolic activation factors, as indicated by the concentration ratio.     

Functional relationship of the cytochrome b to the superoxide-generating oxidase of human neutrophils

A subcellular particulate fraction containing the NADPH-dependent O2.--generating oxidase from stimulated human neutrophils was prepared. This fraction was depleted of certain enzyme markers of primary and secondary granules and was devoid of measurable myeloperoxidase, both enzymatically and spectrally. When prepared from neutrophils which had been previously stimulated with phorbal myristate acetate, this fraction contained cyanide-insensitive, pyridine nucleotide-dependent O2.--generating activity with a specific activity of 260 nmol min-1 mg-1. O2.--generating activity is completely ablated by p-chloromercuribenzoate exposure. Preparations from normal unstimulated neutrophils or stimulated neutrophils from a male patient with chronic granulomatous disease had negligible amounts of this O2.--generating enzymatic activity. The dominant chromophore in this preparation was a b-type cytochrome, the spectral and functional characteristics of which are further described herein. Pyridine nucleotide-dependent reduction of the intrinsic cytochrome b closely parallels O2.- generation in this preparation. Specifically, reduction occurs in preparations from phorbal myristate acetate-stimulated neutrophils and is absent in unstimulated or stimulated p-chloromercuribenzoate-inactivated preparations.     

Effective inhibition of Candida albicans growth by the combination of murine peritoneal neutrophils and activated macrophages.

The effect of leukocytes on the anti-Candida activity of neutrophils was examined. Murine neutrophils which were purified from casein-induced peritoneal cells inhibited the mycelial growth of Candida albicans. This anti-Candida activity of neutrophils was augmented by the addition of spleen cells prepared from mice pretreated with bacterial lipopolysaccharide 3 hr before, but not from non-treated mice. The population in the spleen cells, which enhanced the anti-Candida activity of neutrophils, was plastic-plate adherent, nylon-fiber columns adherent and anti-Mac-1 antigen-positive. These immunological profiles suggested that the enhancing cells are classified to splenic macrophages. Peritoneal-exudated macrophages from mice treated with lipopolysaccharide also augmented the anti-Candida activity of neutrophils. These results suggest that the anti-Candida activity of neutrophils may be upregulated by activated macrophages.     

A requirement of MAPKAPK2 in the uropod localization of PTEN during FMLP-induced neutrophil chemotaxis

The directionality control in chemotaxis is the result of a reciprocal regulation of PI3-kinase and PTEN subcellular localization. MK2(-/-) neutrophils have a directionality loss in fMLP-induced chemotaxis. We found that in polarized WT neutrophils PTEN was localized in the uropod region. However, MK2(-/-) neutrophils or p38 MAPK inhibitor-SB203580-pretreated WT neutrophils showed a disrupted PTEN subcellular localization. Some PTEN was localized at the leading edge of the polarized neutrophils, which may lower the concentration of PI3-kinase lipid product PtdIns(3,4,5)P3 required for directionality sensing. FMLP-stimulated MK2(-/-) neutrophils or SB203580-pretreated WT neutrophils also had disrupted F-actin polarization. F-actin polymerization inhibitor lantrunculin-B disrupted the polarization of PTEN, but not PtdIns(3,4,5)P3. The results suggest that PTEN uropod polarization is F-actin polymerization-dependent and may be through the effect of MK2 on F-actin polarization.     

Effect of neutrophilokines on the immune response in mice

The influence of different peptide fractions obtained from the intact and latex-stimulated neutrophils on the immune response was studied. It was demonstrated that neutrophils after stimulation synthesize the factors activating immune response, the intact neutrophils synthesize the suppressor factors of peptide nature.     

Characteristics of the production of active oxygen species from adherent and non-adherent neutrophils

Our objective was to evaluate the characteristics of the production of AOS from the neutrophils that had adhered to the endothelial cells, fibronectin or polystyrene, using the method of electron paramagnetic resonance (EPR) spin trapping. Neutrophils and endothelial cells were isolated from human venous blood and umbilical veins, respectively. AOS production from neutrophils was not elicited only by adhesion. The stimulation of adherent neutrophils with phorbol myristate acetate (PMA) induced the production of AOS. The production of AOS from adherent neutrophils to endothelial cells, but not to fibronectin or polystyrene, decreased with the interval time between the adhesion and the stimulation by PMA. The amount of AOS produced by the neutrophils adherent to fibronectin or polystyrene was maintained for one hour after stimulation with PMA, whereas that by suspended neutrophils gradually decreased with the time after stimulation. Results indicate that adherent and non-adherent neutrophils exhibit differing time course of AOS production.     

Neurogenic inflammation of the rat trachea: fate of neutrophils that adhere to venules

The goal of this study was to determine whether neutrophils that adhere to the vascular endothelium in association with neurogenic inflammation in the respiratory tract migrate out of the blood vessels or whether they detach and reenter the circulation. We also sought to determine whether the fate of the neutrophils is influenced by neutral endopeptidase (NEP), an enzyme that degrades the tachykinins that produce neurogenic inflammation. Neutrophils in the tracheal mucosa of anesthetized pathogen-free rats were examined 5 min or 4 h after neurogenic inflammation was produced by an injection of capsaicin (100 or 200 micrograms/kg iv). In whole mounts of these tracheae stained histochemically for myeloperoxidase, adherent intravascular neutrophils had a spherical or teardrop (regular) shape and migrating neutrophils had a polarized amoeboid (irregular) shape. The number of regular neutrophils in the tracheae was increased at both times, but the increase at 4 h was only half that present at 5 min. The reduction between 5 min and 4 h was not offset by an appreciable increase in the number of irregular neutrophils, unless NEP was inhibited by phosphoramidon. We interpret these results as indicating that the rapid adherence of neutrophils to the vascular endothelium after an injection of capsaicin is followed by a gradual reentry of the neutrophils into the circulation and comparatively little neutrophil migration. However, when the effect of the stimulus is increased and/or prolonged by inhibition of NEP, some of the adherent neutrophils migrate out of the vessels. Thus the activity of NEP can regulate both the magnitude of the neutrophil adherence and the fate of the adherent cells.     

The mechanisms of the neutrophil suppression of creatine kinase and aspartate aminotransferase activities in rat skeletal muscles]

Abdominal neutrophils effect on rat skeletal muscle m. soleus was investigated in vitro. The incubation was carried out in Hanks balanced solution within 24 hrs. It was a release of proteins from m. soleus 1 hr later. Creatine kinase (CK) and aspartate aminotransferase (AAT) activities increase was detected in incubation medium. The neutrophils released their proteins quicker than muscles. A dramatic inhibition of CK and AAT activities took place during coincubation of m. soleus and neutrophils. Zymosan-activated cells had a higher inhibition potency in comparison to nonactivated neutrophils. Analysis of proteinase and myeloperoxidase activities in incubation medium has given evidence that CK and AAT inhibition by non-activated neutrophils mainly depends on cell-secreted proteinases. Zymosan-activated neutrophil inhibition of CK and AAT consists of proteinases and myeloperoxidase effects. AAT appeared to be more resistant than CK to the damage by neutrophils. The used approach failed to demonstrate the direct damage effect of neutrophils on m. soleus, but the described enzyme inhibition mechanism can take place in vivo during leukocyte infiltration of skeletal muscles after intensive muscular activity.     

Tumor-associated neutrophils suppress antitumor immunity of NK cells through the PD-L1/PD-1 axis

Studies have begun to emerge showing the protumor effects of tumor-associated neutrophils (TANs) in tumorigenesis, which may involve dysfunction of NK cells. However, the mechanism through which these rebellious neutrophils modulate NK cell immunity in tumor-bearing state remains unclear. In the present study, we demonstrate that neutrophils can impair the cytotoxicity and infiltration capability of NK cells, and downregulate CCR1 resulting in the weakened infiltration capability of NK cells. Moreover, neutrophils can decrease the responsiveness of NK-activating receptors, NKp46 and NKG2D. Mechanistically, enhanced PD-L1 on neutrophils and PD-1 on NK cells, and subsequent PD-L1/PD-1 interactions were the main mechanisms determining the suppression of neutrophils in NK cell immunity. G-CSF/STAT3 pathway was responsible for PD-L1 upregulation on neutrophils, while IL-18 was essential for PD-1 enhancement on NK cells. The crosstalk between neutrophils and NK cells was cell-cell interaction-dependent. These findings suggest that neutrophils can suppress the antitumor immunity of NK cells in tumor-bearing status through the PD-L1/PD-1 axis, highlighting the importance of PD-L1/PD-1 in the inhibitory effect of neutrophils on NK cells. Targeting G-CSF/STAT3 and IL-18 signaling pathway may be potential strategies to inhibit residual tumor in tumor therapy.     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

Arachidonate metabolite(s) increase the permeability of the plasma membrane of the neutrophils to calcium

The relationship between arachidonate metabolism and stimulated calcium fluxes in rabbit peritoneal neutrophils has been investigated. The addition of arachidonate to the neutrophils was found to cause a rapid and significant increase in the permeability of the plasma membrane to calcium. This effect is specific to calcium, concentration dependent and sensitive to inhibitors of the lipoxygenase mediated metabolic pathway(s). These results strongly suggest that arachidonate metabolites are directly involved in the mechanisms underlying calcium gating in the neutrophils.     

Host defense function in neutrophils from the American bison (Bison bison)

Selected host defense functions of neutrophils isolated from American bison (Bison bison) were characterized and compared with those of cattle (Bos taurus). Bison neutrophils had a robust chemotactic response to both IL-8 and LTB(4), with maximal responses occurring at 10(-7) M (IL-8) and 10(-8) M (LTB(4)). The magnitude of the chemotactic response to IL-8 was similar in bison and bovine neutrophils (except at 10(-7) M IL-8, where bison had a stronger response). In response to LTB(4), bison neutrophils had a much stronger chemotaxis at both 10(-8) and 10(-7) M than did bovine cells. Production of reactive oxygen species (ROS) in response to phorbol myristate acetate (PMA) and opsonized zymosan (OpZ) was similar between bison and bovine neutrophils. However, the production of ROS in bison neutrophils stimulated with OpZ was primarily intracellular, while extracellular release of ROS was evident in bovine neutrophils stimulated with OpZ. Like bovine neutrophils, bison neutrophils did not generate a respiratory burst in response to fMLF. Granules prepared from bison neutrophils had potent direct killing action on the Gram-negative bacteria Escherichia coli but failed to kill the Gram-positive bacteria Staphylococcus aureus and, at intermediate doses, actually had a permissive effect for this bacteria. Thus, bison neutrophils have potent host defense capabilities similar in quality to those of bovine neutrophils; however, unique differences are present, which may allow bison neutrophils to respond to the distinct immunological challenges that bison encounter.     

Stimulated chemotactic response in neutrophils from Trichinella pseudospiralis-infected mice and the neutrophilotactic potential of Trichinella extracts.

During infection with Trichinella pseudospiralis a strong neutrophil response is evident in the peripheral circulation of the mouse. This study compared the chemotactic response of neutrophils from uninfected, T. pseudospiralis-infected and Trichinella spiralis-infected mice to extracts from adult worms, newborn larvae and muscle-stage larvae of both species of parasite. The chemotactic response of neutrophils from T. pseudospiralis-infected mice to Zymosan-activated mouse serum (ZAMS) was significantly greater than that seen with neutrophils from either uninfected or T. spiralis-infected mice. Unstimulated chemotactic response of neutrophils from these three groups of animals to medium alone was similar. The chemotactic response of neutrophils from the three groups of animals was unaffected by either the concentration or source of serum. The chemotactic response of neutrophils from T. pseudospiralis-infected mice was significantly greater than that observed with cells from uninfected or T. spiralis-infected mice. Among parasite extracts, those from newborn larvae displayed the strongest chemotactic potential for neutrophils. Extracts from muscle larvae of T. spiralis and T. pseudospiralis and extracts of T. spiralis adult worms showed the weakest attraction for neutrophils. Extracts from adult T. pseudospiralis and from newborn larvae of both species elevated the chemotactic response of uninfected mouse neutrophils to a significantly greater level than that seen with ZAMS alone, while a significant reduction in this response was evident only when ZAMS was presented to neutrophils with 500 micrograms of extract from muscle larvae of T. pseudospiralis or T. spiralis.(ABSTRACT TRUNCATED AT 250 WORDS)     

6-formylpterin intracellularly generates hydrogen peroxide and restores the impaired bactericidal activity of human neutrophils.

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H(2)O(2)) occurred. The H(2)O(2) generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H(2)O(2) independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.     

Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane.

Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.     

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.     

Expression of the estrogen receptor in blood neutrophils of dairy cows during the periparturient period

During the period around parturition, cows experience an increased susceptibility to inflammatory disorders in the mammary gland and uterus. This increased susceptibility has been correlated with a decreased functionality of neutrophils, major components in the innate immune defence. As sex steroid levels vary extensively in the period around parturition, an influence of these changes on the functionality of neutrophils has been suggested. Indeed, it has been shown that 17beta-estradiol affects some functions of bovine neutrophils. In spite of these observations, receptors for 17beta-estradiol have not yet been demonstrated in these cells. The investigation of the presence of estrogen receptors in bovine neutrophils was therefore the main objective of this study. The expression of estrogen receptors was evaluated at the protein level by flow cytometry, and at the mRNA level by polymerase chain reaction. A clear positive signal was obtained using flow cytometry for the estrogen receptor protein in bovine neutrophils. Further discrimination between the estrogen receptor subtypes alpha and beta revealed the expression of the estrogen receptor beta, whereas for the estrogen receptor alpha no reproducible positive signal could be obtained with the available antibodies. Both subtypes were found at the mRNA level. Subsequently, the estrogen receptor protein expression level in neutrophils obtained from cows in early lactation was compared with those from cows in late pregnancy. Additionally, the influence of endogenous 17beta-estradiol and progesterone levels was assessed. No difference was found for the estrogen receptor protein expression in neutrophils from cows in early lactation compared with late gestation neither were the endogenous 17beta-estradiol and progesterone levels correlated with the protein expression.