Fecal calcium levels of bird nestlings as a potential indicator of species-specific metal sensitivity.

Sensitivity of bird species to environmental metal pollution varies but there is currently no general framework to predict species-specific sensitivity. Such information would be valuable from a conservation point-of-view. Calcium (Ca) has antagonistic effects on metal toxicity and studies with some common model species show that low dietary and circulating calcium (Ca) levels indicate higher sensitivity to harmful effects of toxic metals. Here we measured fecal Ca and five other macroelement (potassium K, magnesium Mg, sodium Na, phosphorus P, sulphur S) concentrations as proxies for dietary levels in 66 bird species to better understand their interspecific variation and potential use as an indicator of metal sensitivity in a wider range of species (the main analyses include 39 species). We found marked interspecific differences in fecal Ca concentration, which correlated positively with Mg and negatively with Na, P and S levels. Lowest Ca concentrations were found in insectivorous species and especially aerial foragers, such as swifts (Apodidae) and swallows (Hirundinidae). Instead, ground foraging species like starlings (Sturnidae), sparrows (Passeridae), cranes (Gruidae) and larks (Alaudidae) showed relatively high fecal Ca levels. Independent of phylogeny, insectivorous diet and aerial foraging seem to indicate low Ca levels and potential sensitivity to toxic metals. Our results, together with information published on fecal Ca levels and toxic metal impacts, suggest that fecal Ca levels are a promising new tool to evaluate potential metal-sensitivity of birds, and we encourage gathering such information in other bird species. Information on the effects of metals on breeding parameters in a wider range of bird species would also help in ranking species by their sensitivity to metal pollution.

Topical cis-urocanic acid attenuates oedema and erythema in acute and subacute skin inflammation in the mouse.

BACKGROUND: cis-Urocanic acid (cis-UCA) is an endogenous immunosuppressive molecule of the epidermis. OBJECTIVES: We investigated the effects of topical cis-UCA creams (2·5% and 5%) in acute and subacute mouse models of skin inflammation. METHODS: Acute skin irritation was induced by applying dimethyl sulphoxide (DMSO) on the earlobe of CD-1 mice. Topical cis-UCA, hydrocortisone (1%) or tacrolimus (0·1%) were applied 10 min later. In another model, subacute inflammation was provoked and maintained by three applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the ears of NMRI mice on days 1, 2 and 4. The test products were applied topically twice a day during 6 days. RESULTS: In the acute DMSO model, cis-UCA creams suppressed ear swelling at 1 h significantly more efficiently than hydrocortisone (P < 0·01) and tacrolimus (P < 0·001). Ear swelling was significantly inhibited by cis-UCA (P < 0·001) in the subacute TPA model as well. The 5% cream also decreased erythema, whereas tacrolimus enhanced skin reddening. Treatments with cis-UCA did not affect TPA-induced infiltration of neutrophils to the skin. In contrast to hydrocortisone, cis-UCA did not reduce epidermal thickness. CONCLUSIONS: The results suggest that cis-UCA - unlike hydrocortisone and tacrolimus - is efficient in both acute and subacute skin inflammation, attenuating skin oedema and erythema. Topical drug therapy with cis-UCA may provide a safe and effective drug treatment modality in inflammatory skin disorders.

Cis-urocanic acid suppresses UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal and conjunctival epithelial cells in vitro.

PURPOSE: Urocanic acid (UCA) is a major ultraviolet (UV)-absorbing endogenous chromophore in the epidermis and is also an efficacious immunosuppressant. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in ocular surface cell cultures exposed to UV-B irradiation. METHODS: Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCECs) were incubated with 10, 100, 1,000, and 5,000 microg/ml cis-UCA with and without a single UV-B irradiation dose. The concentrations of IL-1beta, IL-6, IL-8, and TNF-alpha in the culture medium and caspase-3 activity in the cell extract sampled were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was measured by the colorimetric MTT (3-(4,5-dimethyldiazol- 2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: UV-B irradiation multiplied interleukin IL-6 and IL-8 secretion levels in HCE-2 cells and HCECs as analyzed with ELISA. Cell viability as measured by the MTT assay declined by 30%-50% in HCE-2 cells and by 20%-40% in HCECs after UV-B irradiation. Moreover, UV-B increased caspase-3 activity in both cell types as analyzed with ELISA. Treatment with 100 microg/ml cis-UCA completely suppressed IL-6 and IL-8 secretion, decreased caspase-3 activity, and improved cell viability against UV-B irradiation. No significant effects on IL-6 or IL-8 secretion, caspase-3 activity, or viability of the non-irradiated cells were observed with 100 microg/ml cis-UCA in both cell types. The 5,000 microg/ml concentration was toxic. CONCLUSIONS: These findings indicate that cis-UCA may represent a promising anti-inflammatory and cytoprotective treatment option to suppress UV-B-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.

Effect of cis-urocanic acid on bovine neutrophil generation of reactive oxygen species.

Neutrophils play a fundamental role in the host innate immune response during mastitis and other bacterial-mediated diseases of cattle. One of the critical mechanisms by which neutrophils contribute to host innate immune defenses is through their ability to phagocytose and kill bacteria. The ability of neutrophils to kill bacteria is mediated through the generation of reactive oxygen species (ROS). However, the extracellular release of ROS can be deleterious to the host because ROS induce tissue injury. Thus, in diseases such as mastitis that are accompanied by the influx of neutrophils, the generation of large quantities of ROS may result in significant injury to the mammary epithelium. cis-Urocanic acid (cis-UCA), which is formed from the UV photoisomerization of the trans isoform found naturally in human and animal skin, is an immunosuppressive molecule with anti-inflammatory properties. Little is known about the effect of cis-UCA on neutrophils, although one report demonstrated that it inhibits human neutrophil respiratory burst activity. However, the nature of this inhibition remains unknown. Because of the potential therapeutic use that a molecule such as cis-UCA may have in blocking excessive respiratory burst activity that may be deleterious to the host, the ability of cis-UCA to inhibit bovine neutrophil production of ROS was studied. Further, because neutrophil generation of ROS is necessary for optimal neutrophil bactericidal activity, a response which is critical for the host innate immune defense against infection, the effects of cis-UCA on bovine neutrophil phagocytosis and bacterial killing were assayed. cis-Urocanic acid dose-dependently inhibited the respiratory burst activity of bovine neutrophils as measured by luminol chemiluminescence. Subsequently, the effect of cis-UCA on the production of specific oxygen radicals was investigated using more selective assays. Using 2 distinct assays, we established that cis-UCA inhibited the generation of extracellular superoxide. In contrast, cis-UCA had no effect on the generation of intracellular levels of superoxide or other ROS. At concentrations that inhibited generation of extracellular superoxide, bovine neutrophil phagocytosis and bacterial activity remained intact. Together, these data suggest that cis-UCA inhibits the tissue-damaging generation of extracellular ROS while preserving neutrophil bactericidal activity.

Effects of prolonged EGF treatment on phospholipid turnover and DAG formation in murine keratinocytes.

In murine keratinocytes, the cellular diacylglycerol (DAG) content was considerably elevated following a 48-h exposure to epidermal growth factor (EGF), while formation of inositol phosphates (InsP) was not stimulated. A similar loss of InsP production upon stimulation of keratinocytes with 1.4 mM Ca2+ was seen after pretreatment with R59022, a DAG kinase inhibitor. These data suggest that accumulated endogenous DAG has an inhibitory feedback effect on PLC activity. To elucidate the possible phospholipid source of elevated DAG in keratinocytes, cells were first labeled with [3H]-choline and then exposed to EGF for 24 h or TPA, a protein kinase C activator, for 8 h. As expected, TPA increased [3H]-choline release into the culture medium, whereas EGF decreased the release, suggesting that EGF treatment does not result in sustained stimulation of phosphatidylcholine turnover. The release of [14C]-dihomo-gamma-linolenic acid (DHGLA), predominately bound to the 2-positions of phospholipids, was also stimulated by 8 h of TPA treatment but not by 24 h of EGF treatment. The distribution of DHGLA in various phospholipid subclasses was not influenced by EGF. These results indicate that prolonged EGF treatment does not markedly activate phospholipid A2 (PLA2) or lysophospholipase, and that the DAG accumulation after prolonged EGF exposure is apparently not associated with stimulated breakdown of any specific lipid pool. It is concluded that changes in keratinocyte lipid turnover induced by prolonged EGF treatment differ from those associated with short-term EGF exposure.

Biphasic formation of inositol phosphates in opsonized zymosan-stimulated human neutrophils.

Stimulation by serum-opsonized zymosan (SOZ) typically causes a biphasic rise in the cytosolic free Ca2+ concentration ([Ca2+]i) of human neutrophils. It consists of an initial slow Ca2+ release from internal pools lasting for 60 s, followed by a rapid but sustained influx of Ca2+. It was the aim of this study to elucidate the underlying mechanism of this atypical Ca2+ response. For this reason we analysed the production of inositol phosphates (InsPs) in myo-[3H]inositol labelled cells. Stimulation by SOZ within 10 s transiently elevated inositol trisphosphate (InsP3) by 1.50-fold. This response was followed by a second, more sustained 1.55-fold rise in InsP3 by 90 s. A similar, biphasic pattern of inositol tetrakisphosphate (InsP4) formation with 1.15- and 1.35-fold increases, respectively, was observed. The SOZ-induced formation of InsP3 was unaffected by the removal of extracellular Ca2+ by 1.4 mM EGTA. In contrast, the early accumulation of InsP4 was stronger and more prolonged and no second rise over the baseline level was seen in the absence of extracellular Ca2+. Under these conditions, the sudden exposure of Fura-2 AM loaded, SOZ-stimulated neutrophils to extracellular Ca2+ at a time point where InsP4 was the predominant InsP resulted in a marked increase in [Ca2+]i. Recalcification at a time point when InsP3 was the major InsP had no effect on [Ca2+]i. These findings suggest that in SOZ-stimulated neutrophils (1) the transient, first accumulation of InsP3 mediates the slow Ca2+ release from internal pools, and (2) the second, more pronounced formation of InsP4 triggers the Ca2+ influx.(ABSTRACT TRUNCATED AT 400 WORDS)

The up- and down-modulation of immunoglobulin G Fc receptors and complement receptors on activated human neutrophils depends on the nature of activator.

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.

Histamine modulation of Ca2+ homeostasis in human neutrophils.

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.

Systemic suppression of human peripheral blood phagocytic leukocytes after whole-body UVB irradiation.

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.

Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand.

A new 96-well microtiter plate, time-resolved fluorometric assay was developed to measure leukocyte adhesion in vitro. The assay is based on loading leukocytes with a fluorescence enhancing ligand 2,2':6', 2"-terpyridine-6,6"-dicarboxylic acid (TDA), which in its acetoxymethyl ester form readily diffuses through the cell membrane. After hydrolysis by nonspecific intracellular esterases, the impermeable TDA accumulates inside the cells. When the TDA-labeled adherent leukocytes are lysed, the ligand is released and reacts with europium present in the lysis solution to produce a highly fluorescent and stable chelate. The fluorescence signal can be measured by time-resolved fluorometry and correlates directly with the number of adherent cells. In this study, we have optimized both the TDA-labeling and adhesion assay conditions in isolated human neutrophils. Furthermore, we have compared the assay with a traditional microscopic counting method. This time-resolved fluorometric assay provides a rapid, reproducible and convenient method for the routine analysis of leukocyte adhesion.

Human recombinant GM-CSF selectively primes receptor mediated respiratory burst of neutrophils in vitro.

The influence of human recombinant granulocyte-macrophage colony-stimulating factor (rH GM-CSF) on respiratory burst response of isolated human neutrophils was examined. Preincubation of cells with rH GM-CSF significantly increased the respiratory burst in response to formyl-methionyl-leucyl-phenylalanine (FMLP), measured by luminol-dependent chemiluminescence (CL) assay. This priming effect of rH GM-CSF was independent of extracellular Ca2+ and Mg2+. On the other hand, the pretreatment of cells with rH GM-CSF could not enhance the neutrophil CL responses to unopsonized, serum complement-opsonized or immunoglobulin G (IgG)-opsonized zymosan particles. rH GM-CSF directly induced a weak CL signal in neutrophils. This signal, however, was abolished when extracellular Ca2+ and Mg2+ were removed. Exposure to rH GM-CSF caused a divalent cation-dependent up-regulation of complement receptors (CR1 and CR3) on neutrophil cell surface, while the expression of IgG Fc-receptors (FcRII and FcRIII) was not markedly changed by rH GM-CSF. The results indicate that rH GM-CSF primes FMLP-induced CL but not zymosan particle-induced respiratory burst in human neutrophils. It is hypothesized that the reason for the different sensitivity of FMLP-receptors and receptors to zymosan particles to rH GM-CSF priming may lie in differences in the signal-transduction pathways of these receptor types.

The reappearance of 10 differentiation antigens on peripheral blood lymphocytes after allogeneic bone marrow transplantation.

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.

Neutrophils are responsible for the reappearance of chemiluminescence after allogeneic bone marrow transplantation.

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.

Antioxidant enzymes are elevated in dimethylbenz[a]anthracene-induced neoplastic murine keratinocytes containing an active rasHa oncogene.

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.

A review of studies on the effects of ultraviolet irradiation on the resistance to infections: evidence from rodent infection models and verification by experimental and observational human studies.

Recent studies on the immunosuppressive effects of ultraviolet radiation (UVR) and the related resistance to infections in rodents and humans are presented. The waveband dependency of trans-to-cis isomerisation of urocanic acid in the stratum corneum and the role of DNA damage in UVR-induced erythema and immunosuppression were investigated to further elucidate the underlying mechanisms. Furthermore, human experimental studies on UVR-induced immunomodulation were performed. It appeared that the doses needed to suppress various immune parameters in humans (e.g. NK activity, contact hypersensitivity) were higher than those needed in experiments in rodents. Still, extrapolation of experimental animal data to the human situation showed that UVR may impair the resistance to different systemic infections at relevant outdoor doses. In observational human studies we aimed to substantiate the relevance of UVR for infections in humans. It was shown that sunny season was associated with a slightly retarded but clinically non-relevant antibody response to hepatitis B vaccination. Furthermore, sunny season appeared to be associated with a small decline in the number of CD4+ T-helper cells in a cohort of HIV-infected persons and a higher recurrence of herpes simplex and herpes zoster in a cohort of renal transplant recipients. However, in a study among young children a higher exposure to solar UVR was associated with a lower occurrence of upper respiratory tract symptoms. As disentangling the effects of UVR from other relevant factors is often impossible in observational studies, concise quantitative risk estimations for the human situation cannot be given at present.

Urocanic acid suppresses the activation of human neutrophils in vitro.

Exposure to ultraviolet (UV) light impairs the function of inflammatory cells. Urocanic acid (UCA) in an stratum corneum has been suggested as a mediator in the immunosuppression of lymphoid cells detected after irradiation with UVB (UV wavelengths 280-320 nm). In this study, we examined the effects of the two UCA isomers, trans and cis UCA on human polymorphonuclear leukocytes, neutrophils. It was found that treatment of cells with either trans of cis UCA isomers inhibited the opsonized zymosan-induced respiratory burst activity, measured with luminol-enhanced chemiluminescence assay. Both isomers were also able to partially block the up-regulation of complement receptors 1 (CR1; CD35) and 3 (CR3; CD11b/ CD18) in N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These results indicate that the isomerization of trans UCA to cis UCA is not essential for the action of UCA on neutrophils. Neither of the UCA isomers were found to induce cyclic AMP (cAMP) formation in 3-isobutyl-1-methylxanthine treated cells, suggesting that the activation of adenylate cyclase cAMP system is not involved in UCA provoked suppression of neutrophils. It is concluded that the function of UCA may be protective, to suppress the activation of human neutrophils in inflamed, sunburned epidermis.

Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles.

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.

Regulation of activation of bovine neutrophils by aggregated immunoglobulin G.

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.

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin.

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.

An automated chemiluminescence test for diagnosis of leukocytospermia.

A new rapid and sensitive chemiluminescence (CL) test was developed for the analysis of leucocytes in semen. Opsonized zymosan induced luminol-dependent CL of diluted (1:500) semen was measured in samples from 64 fertile and infertile men with or without leukocytospermia, using an automated luminometer set-up. The white-blood-cell (WBC) count in semen was determined using a conventional leucocyte-peroxidase staining method. A good linear correlation (r = 0.932) was observed between the seminal white-blood-cell number and the CL response. The coefficients of inter-assay variations for the CL method were 9.6% and 1.8% for semen samples with 0.3 x 10(6) WBC ml-1 and 3.2 x 10(6) WBC ml-1, respectively. The results also suggest that the previously reported inhibitory effect of seminal plasma on CL activity of human phagocytes is due to the quenching of light-producing reactions and that this can be circumvented by using appropriate semen dilution. It is concluded that the simple and high-capacity CL test is an especially convenient method for routine diagnosis of leukocytospermia.