The neutrophil

The association between transportation and the occurrence of the bovine respiratory disease complex (BRDC) has long been recognised. Many hypotheses regarding this association have been declared through the past decades, and it is agreed upon by most researchers that the multiple stressors that calves experience during transportation result in an overall immunosuppression that allows the respiratory tract to be invaded by numerous opportunistic pathogens. Furthermore, the innate immune cells, neutrophils, may be trapped in a paradox whereby their crucial defence and pathogen-killing activities are counteracted by excessive inflammation and tissue damage that may exacerbate disease, including the BRDC. Neutrophilia in response to glucocorticoids has been attributed to an influx of immature neutrophils newly released from the bone marrow, a decrease in neutrophil margination along endothelial walls, and a decrease in neutrophil apoptosis. Several of these explanations have been confirmed by altered expression of genes and proteins important for neutrophil margination and apoptosis.

Albumin, among other molecules, binds and detoxifies endotoxin in healthy people. Oxidative stress leads to protein oxidation and thus to the impaired binding properties of albumin. This property, in combination with increased gut permeability, leads to the appearance of endotoxin in the systemic circulation and to impaired organ function. We hypothesize that these processes occur in the serum of brain-dead organ donors. Endotoxin was determined with an adapted Limulus amoebocyte lysate assay. The albumin fractions and binding capacity were determined by high-performance liquid chromatography (HPLC). FlowCytomix (eBioscience, San Diego, Calif) was used to determine the cytokine levels. Carbonylated proteins (CPs) and myeloperoxidase (MPO) were measured by an enzyme-linked immunosorbent assay (ELISA). Eighty-four brain-dead organ donors were enrolled and categorized by the duration of intensive care unit (ICU) stay. The albumin-binding capacity for dansylsarcosine was reduced in brain-dead patients compared with controls. Endotoxin positivity in 16.7% of donors was associated with decreased binding capacity in donors and worse survival of recipients. The CP and MPO levels of organ donors were significantly higher than in healthy controls. The durations of ICU stay increased albumin oxidation. In addition, interleukin-6 (IL-6), IL-8, IL-10, and IL-1β levels were increased in patients, whereas the interferon-γ (IFN-γ) levels were within the normal range. We conclude that oxidative stress and systemic endotoxemia are present in brain-dead organ donors, which might affect recipient survival. High endotoxin levels might be caused by increased gut permeability and decreased binding capacity of albumin influenced not just by higher albumin oxidation.

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-α in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-α on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-α primed neutrophils for migration to CCL3 via CCR5. TNF-α-induced migration was a consequence of the TNF-α-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-α activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-α on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-α unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-α regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-α-mediated inflammatory states.

Stress and its association with altered immune function and incidence of respiratory diseases in cattle have lead to concerns over animal health and welfare during truck transportation. Previously, bulls subjected to transportation stress displayed altered expression of candidate neutrophil genes, warranting a broader investigation of the neutrophil transcriptome and possible associations with fluctuations in circulating steroid hormones. In the current study, blood was collected from six Belgian Blue × Friesian bulls at −24, 0, 4.5, 9.75, 14.25, 24, and 48 h relative to initiation of 9 h of truck transportation. Plasma concentrations of cortisol, dehydroepiandrosterone (DHEA), progesterone, and testosterone were measured; cortisol:DHEA ratios were computed. Neutrophil gene expression was monitored by microarray analysis using bovine immunobiology (BOTL-5) microarrays. Eighty-eight genes were identified as being differentially expressed at P < 0.05. Putatively affected genes were grouped into ontological clusters; those of greatest interest for qRT-PCR validation were involved in immune response, apoptosis, wound healing, and several of currently unknown function. Confirmed gene expression changes supported the dramatic effects of transportation stress on the bovine neutrophil transcriptome. Temporal correlations between gene expression profiles and circulating total leukocyte and neutrophil counts were apparent. However, few relationships between gene expression and plasma steroid profiles were detected, possibly due to the biological time-lag between these variables not captured by the blood collection schedule. Further investigation into the factors underlying neutrophil gene expression changes and validations at the protein and cell behavior levels will lead to a better understanding of altered innate immunity in cattle during transportation stress.

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24 h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.

Neutrophils undergo rapid Fas-mediated apoptosis during in vitro culture. The purpose of this study was to investigate the effects of surgical stress upon the Fas-mediated apoptotic response in circulating neutrophils.

Blood samples were drawn from eight patients with a mandibular prognathism, and who had undergone a bilateral sagittal split ramus osteotomy, at 2 days before, and at 1 and 5 days after surgery. The circulating neutrophils in each blood sample were then evaluated for their susceptibility to Fas-mediated apoptosis in either the presence or the absence of autogenous plasma.

Fas-induced apoptosis in the neutrophils of these surgically treated patients was found to be slightly accelerated at 1 day postoperatively in the presence of FBS, compared with 2 days preoperatively and 5 days postoperatively. However, we obtained different results for these experiments in the presence of autogenous plasma. The Fas-induced apoptotic response levels in the neutrophils at day 1 postsurgery following exposure to autogenous plasma were significantly suppressed compared with the levels at both 2 days preoperatively and 5 days postoperatively. The Fas expression levels on the cell surface of the neutrophils were not altered, but the levels of soluble Fas (sFas) in the plasma were reduced to almost inverse levels during the postoperative periods. The levels of granulocyte-macrophage colony-stimulating factor, interleukin-6, and interleukin-8 levels in the plasma were also markedly raised in the plasma from each of these patients at 1 day postoperatively. However, the anti-apoptotic effects of the plasma on the Fas-mediated neutrophil apoptosis were not influenced by the addition of their neutralizing antibodies for these cytokines. The suppressive effects of postoperative plasma on Fas-mediated neutrophil apoptosis were blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, LY294002, and wortmannin. Additionally, these effects were also abrogated by the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, but not by the p38 mitogen-activated protein kinase inhibitor, SB203580.

The increase in sFas levels in the plasma of patients with acute inflammation may lead to the inhibition of Fas-mediated neutrophil apoptosis. Moreover, the activation of the PI 3-K and ERK signaling-dependent pathways may, in part, also contribute to the down-regulation of the Fas-mediated apoptotic response in neutrophils.