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Objective: To evaluate the effects of cis-urocanic acid (cis-UCA) on mammary gland (MG) inflammation and injury associated with Escherichia coli-induced mastitis in dairy cows., Animals: 12 lactating dairy cows (36 MGs)., Procedures: At 7-week intervals, a different MG in each cow was experimentally inoculated with E coli. At 6-hour intervals from 6 to 36 hours after inoculation, the inoculated MG in each cow was infused with 40 mL of saline (0.9% NaCl) solution, 12.5mM cis-UCA, or 25mM cis-UCA (4 cows/group); ultimately, each cow received each treatment. Immediately prior to and at various time points after inoculation and treatment, milk samples were collected. Bacterial CFUs, somatic cell counts (SCCs), N-acetyl-beta-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) activities, and concentrations of bovine serum albumin, tumor necrosis factor-alpha, and cis-UCA were quantified in each milk sample. Results-Compared with findings in saline solution-treated MGs, NAGase and LDH activities in milk samples from cis-UCA-treated MGs were lower. Cis-UCA had no effect on milk SCCs and milk concentrations of bovine serum albumin and tumor necrosis factor-alpha. Furthermore, cis-UCA had no adverse effect on bacterial clearance; CFUs of E coli in MGs treated with saline solution or cis-UCA were equivalent., Conclusions and Clinical Relevance: In cows, milk NAGase and LDH activities were both lower in E coli-infected MGs infused with cis-UCA than in those infused with saline solution, which suggests that cis-UCA reduced mastitis-associated tissue damage. Furthermore, these data indicated that therapeutic concentrations of cis-UCA in milk can be achieved via intramammary infusion.

GTPgammaS activates the NADPH oxidase and this activity declines rapidly with time after preexposure to streptolysin O. This was not due to loss of p47(phox), p67(phox), or Rac. To identify the component(s) leaking out of the permeabilized cell responsible for loss of activity, a GTPgammaS-dependent reconstitution assay was established. Neutrophil cytosol was subjected to chromatographic fractionation steps for purification of the minimum fraction required to restore activity. The reconstitution of the GTPgammaS-stimulated activity was dependent on ATP. The inhibitors staurosporine and calphostin C greatly reduced the activity in the reconstitution assay, implicating the involvement of a protein kinase C (PKC) pathway. PKC isoforms beta and delta were eliminated as the active factors in the most pure reconstitution fraction. With this novel cell-based reconstitution assay, we have identified the requirement for a protein kinase, or its substrate, for the restoration of GTPgammaS activation of the NADPH oxidase., (Copyright 1999 Academic Press.)

We examined systemic effects of whole-body UVB irradiation on human peripheral blood phagocytes. We found that 24 h after a single erythemal dose of UVB radiation two phagocyte functions, adhesion and phagocytosis, were reduced by 50%. This functional suppression was accompanied by a significant decrease in the expression of complement receptors (CR1 and CR3) and IgG Fc receptors (FcRII and FcRIII). The greatest reduction (47%) was observed in CR3, which is important for both adhesion and phagocytosis. A kinetic analysis showed that both CR1 and CR3 levels started to decrease 15 min after the UVB exposure, reaching the lowest levels at 4.5- and 24-h time points, respectively. The down-modulation of CRs after whole-body UVB exposure was not due to a defective receptor synthesis or translocation from internal stores to plasma membrane because the maximal CR levels in stimulated cells were not affected by UVB. No change in the serum soluble ICAM-1 was detected after UVB, which rules out CD1 1b epitope masking by sICAM-1. UVB did not release low-receptor-density myeloid progenitor cells from storage pools into circulation. Interleukin 10, a mediator of UVB-induced immunosuppression, was unable to modulate CR expression in vitro. When seven suberythemal whole-body UVB exposures were given repeatedly within 2 weeks, a significant decrease in CR expression was seen, which was greatest after three irradiations. Our data suggest that an exposure to UVB has systemic effects in humans which, possibly due to the down-modulation of preexisting cell-surface receptors, suppress some important functions of circulating phagocytic cells.

Localized juvenile periodontitis is a destructive form of periodontal inflammatory disease which has its onset at puberty. The etiopathology of the disease is still unclear but neutrophils have been suggested to play a major role both in the production and development of the disorder. About 70% of the patients with localized juvenile periodontitis exhibit neutrophil functional abnormalities, such as decreased chemotaxis and phagocytosis. Interestingly, it has been frequently reported that the same hypoactive cells show an enhanced respiratory burst response and increased adhesion. Several possible mechanisms explaining neutrophil anomalies in localized juvenile periodontitis have been proposed. These include the presence of soluble serum factors capable of modulating neutrophil function, altered cell-surface receptor expression and/or function, and a change in the post-receptor signaling events. Recently, a growing evidence has accumulated showing that the diacylglycerol metabolism could be altered in neutrophils from patients with localized juvenile periodontitis. This change, which may be due to a defect in a major diacylglycerol metabolizing enzyme, diacylglycerol kinase, results in enhanced accumulation of diacylglycerol in activated cells. Because diacylglycerol is an endogenous activator of protein kinase C, the increased and prolonged generation of diacylglycerol could lead to abnormal pattern of protein kinase C-regulated neutrophil functions, explaining the parallel hypo- and hyperactivities.

Deficiency of omithine-delta-aminotransferase (OAT) causes gyrate atrophy of the choroid and retina with hyperornithinemia (GA; McKusick 258870), a progressive autosomal recessive chorioretinal degeneration leading to early blindness. As residual enzyme activity may vary in different mutations of the OAT gene and explain individual variations in disease progression, a sensitive HPLC modification of the OAT assay in lymphocytes was developed, based on measurement of the dihydroquinozolinium reaction product. The OAT activities (ranges) of 43 Finnish GA patients with mutations L402P/L402P, R180T/L402P, N89K/ L402P, and L402P/x (x = previously unknown allele), were <1-10, <1-13, <1-17, and <1 pmol x min(-1) mg protein(-1), respectively. The OAT activities (mean+/-SD) of nine L402P/ wild heterozygotes were 70+/-50 (range 33-193), and those of 15 healthy control subjects 184+/-60 (range 85-291) pmol x min(-1) mg protein(-1). This lymphocyte assay is an easy, rapid, and sensitive method for reliable recognition of GA homozygotes. OAT mutations of the Finnish patients show similar residual enzyme activity in the lymphocytes. OAT activities in the L402P heterozygotes and healthy control subjects overlap, suggesting that, for reliable carrier detection, the OAT alleles have to be studied. However, as all OAT mutations are not known, direct measurement of enzyme activity has a role in heterozygote identification and possibly also in prenatal diagnosis of GA.

Objective: To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils., Animals: 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources., Procedure: Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils., Results: MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes., Conclusion: MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells.

The diacylglycerol (DAG) kinase activity was determined in crude cytosol and membrane fractions in human peripheral blood lymphocytes and neutrophils. In neutrophils the basal lipid kinase activity was identical in the two subcellular fractions whereas in lymphocytes the basal enzyme activity was 1.6-fold higher in the membrane fraction. In general, the DAG kinase activity in lymphocyte fractions was 10- to 20-fold higher than in neutrophil fractions. When lymphocytes were stimulated with phytohaemagglutinin for 4 hours, a significant decrease in the DAG kinase activity in the membrane fraction was detected. By contrast, a 30-min activation with N-formyl-methionyl-leucyl-phenylalanine markedly increased the lipid kinase activity in both subcellular fractions in neutrophils. This activity was partially inhibited by the compound R59022. These results suggest that the DAG kinase is widely distributed in human leukocytes, but the enzyme's activity and regulation may vary in different leukocyte types.

Objectives: To investigate the role of extracellular Ca2+ and Mg2+ in aggregated IgG (aIgG)-mediated cellular activation, and to determine how aIgG-induced activation is coupled to Ca2+ homeostasis in bovine neutrophils., Sample Population: 4 clinically normal, lactating Holstein cows, in their second lactation, which ranged between 60 and 150 days., Procedure: aIgG was prepared by heating bovine IgG, and C5a was obtained by activating fetal bovine serum with zymosan. Luminol-amplified chemiluminescence (CL) of isolated neutrophils was measured in the presence of aIgG or phorbol 12-myristate 13-acetate (PMA). The reaction mixture contained either Hanks' balanced salt solution or Ca(2+)- and Mg(2+)-free Hanks' balanced salt solution. Binding of aIgG to neutrophils was measured by flow cytometry after incubation with fluorescein isothiocyanate-conjugated second antibody. Intracellular-free concentration [Ca2+]i was measured in a fluorescence spectrofluorometer after incubation of neutrophils, loaded with the fluorescent dye fura-2 acetoxymethyl ester, with either aIgG or C5a., Results: In a Ca(2+)- and Mg(2+)-containing reaction mixture, aIgG induced strong CL responses, whereas removal of extracellular divalent cations almost abolished the respiratory burst activity. The CL emission on stimulation with PMA was independent of extracellular Ca2+ and Mg2+. Examination of cells by flow cytometry after incubation with aIgG indicated that the binding of aIgG was identical in the presence and absence of extracellular Ca2+ and Mg2+. No increase in [Ca2+]i was seen in fura-2 acetoxymethylester-loaded neutrophils after stimulation with aIgG. C5a induced a typical transient increase in [Ca2+]i., Conclusions: aIgG-induced activation of bovine neutrophils is highly dependent on presence of extracellular divalent cations. This dependency is not caused by the need of divalent cations for binding of aIgG by neutrophils or because the influx of Ca2+ from the extracellular space is an integral component of aIgG-mediated activation pathway. Because need for extracellular Ca2+ and Mg2+ could be partially circumvented by pretreating neutrophils with PMA, it is possible that this activation pathway may involve a protein kinase C, which is not directly coupled to receptors for aIgG.

Antioxidant enzyme activities and peroxidation potential were measured in primary mouse keratinocytes and neoplastic keratinocytes containing an active rasHa oncogene. In neoplastic cell lines, SP-1 and 308, the activities of Cu, Zn-superoxide dismutase, catalase, and glutathione transferase were significantly elevated. The peroxidation potential was lower in cell homogenates prepared from neoplastic keratinocytes than in those prepared from normal keratinocytes. Consistently, the neoplastic 308 cell line was found to be more resistant than the normal keratinocytes to cytotoxicity induced by UV-B irradiation. The present study suggests that the enhanced antioxidant defense system protects the initiated cells from UV-B-induced oxidative stress, and that the enhanced enzymic antioxidant defense system is potentially a mechanism favoring the selective growth of neoplastic keratinocytes.

The influence of histamine on the basal intracellular free Ca2+ concentration ([Ca2+]i) and agonist-induced increases of [Ca2+]i was studied in Fura-2-loaded neutrophils. Histamine was unable to change the basal [Ca2+]i at concentrations (10(-6)-10(-4) M) that have been shown to cause a rapid increase in [Ca2+]i in a variety of cell types. Histamine, in contrast, was found to inhibit dose-dependently the rise in [Ca2+]i induced by two neutrophil receptor agonists, N-formylmethionyl-leucylphenylalanine (fMLP) and serum-opsonized zymosan particles. The histamine inhibition was shown to be specific for H2 receptor activation by blocking experiments with selective H1 and H2 receptor antagonists. In the absence of extracellular Ca2+, histamine failed to inhibit the agonist-induced rise in [Ca2+]i, indicating that histamine does not affect the release of Ca2+ from internal pools. Forskolin, which mimics the biochemical effects of H2 receptor activation by directly stimulating adenylate cyclase, also decreased the Ca2+ transients induced by receptor agonists. Similarly, 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, reduced the Ca2+ response of activated neutrophils. These data suggest that in human neutrophils (1) no functional H1 receptors are present or alternatively H1 receptors are not coupled to cellular Ca2+ metabolism, and (2) H2 receptors modulate the receptor-triggered Ca2+ flux via the cAMP second messenger system.

Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (CL) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong CL responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the CL reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced CL reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.

Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.

The effects of differently opsonized zymosan particles, acting solely at Fc-gamma or at complement receptors or at both, on the level of intracellular calcium ([Ca2+]i) in human neutrophils were studied. A biphasic, long-lasting increase in [Ca2+]i was seen in response to IgG-, C3- and fresh serum-opsonized zymosan particles in the presence of extracellular Ca2+. Unopsonized zymosan, acting mainly at CR3 failed to elevate [Ca2+]i. Addition of 1.4 mM EGTA reduced but did not abolish the rise in [Ca2+]i triggered by opsonized zymosan, indicating that Ca2+ is released from intracellular stores. EGTA changed also the kinetic patterns of Ca(2+)-responses possibly by indirectly affecting the extrusion of Ca2+ in neutrophils.

Human neutrophils were activated with soluble stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or ionophore A23187, and with opsonized particles, zymosan or Streptococcus pneumoniae bacteria. Monoclonal antibodies and flow cytometry were used to assess the expression of Fc-gamma receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3). The role of extracellular calcium and magnesium in the modulation of receptor expression was also examined. The low-level expression of FcRI was not affected by any activator tested. fMLP and A23187 did not alter the expression of FcRII, whereas a significant, Ca(2+)- and Mg(2+)-independent down-modulation was observed upon activation with opsonized particles. All activators clearly decreased the surface expression of FcRIII in the presence of Ca2+ and Mg2+, probably as a consequence of shedding of the phosphatidylinositol-glycan-anchored receptor protein. The removal of calcium and magnesium blocked the shedding of FcRIII caused by soluble stimuli, whereas it retarded but did not abolish the fall in FcRIII expression when cells were incubated with opsonized particles. This fall was likely due to internalization of the receptor molecules while the shedding was blocked. A rapid increase in CR1 and CR3 expression was seen upon activation with soluble stimuli. The change in CR1 expression was independent of extracellular Ca2+ and Mg2+. The increase in CR3 number required an influx of divalent cations. No total up-modulation of complement receptors occurred when neutrophils were activated with opsonized particles. However, the kinetic analysis revealed a temporary up-modulation that was followed by a down-modulation. The results indicate that the expression of both Fc-gamma and complement receptors on human neutrophils is changed upon activation and that the up- and down-modulation of these receptors depends on the nature of activator. We also suggest that in neutrophils the FcRIII down-modulation is the result of both receptor shedding and internalization, while FcRII is down-modulated by receptor internalization.

Ten monoclonal antibodies and flow cytometry were applied to characterize the recovery of lymphocyte subsets in peripheral blood after allogeneic bone marrow transplantation (BMT). Ten patients were first followed for 150 days (short-term survey) and then analysed 2 years after BMT on average (long-term analysis). Eight of the 10 recipients showed increased relative and absolute numbers of CD8+ cells and reduced numbers of CD4+ cells resulting in an inverse helper/suppressor ratio. In these eight patients the CD8+ cell predominance was long-lasting and still detectable in the long-term analysis. Two patients had a normal helper/suppressor ratio throughout the study but otherwise a similar reconstitution. Despite the slow recovery of CD4+ cells, CD4+ Leu8- and CD4+ CD45RA- helper subsets were in a normal range already on day 30 and their proportions stayed higher than those of CD4+ Leu8+ and CD4+ CD45RA+ helper cells for the whole short-term survey. The number of activated suppressor cells (CD8+ HLA-DR+) increased markedly after BMT. Similarly, in eight patients high numbers of cytotoxic CD8+ CD57+ cells were found from day 50 onwards. An early and sharp rise of NK cells (CD16+, CD56+) was observed in all recipients, and seven recipients also showed an early increase in CD20+ B cells. Later on, normal or slightly elevated numbers of these cells occurred.

The nature of the cells generating the early chemiluminescence (CL) response after allogeneic bone marrow transplantation (BMT) was studied in six patients; five transplanted for acute leukaemia and one for multiple myeloma. Peripheral blood was fractionated into 14 fractions with Percoll narrow range density gradient centrifugation on days +14, +17, +20, and +27 after BMT. The leucocytes were recovered in fractions 7-14 which were analysed for cell morphology and CL response. On days +14 to +20 after BMT the CL response was detected in the fractions containing morphologically mature neutrophils. In one of these fractions the CL response per phagocyte was significantly higher than in the neighbouring fractions although morphologically the cells were similar. The results suggest that the cells responsible for the early CL after BMT are neutrophils and possibly an active subpopulation of neutrophils produced by the marrow graft.