Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets

Monocytes and neutrophils are important players in the innate immune response and cooperate during infection and inflammation. In our study we analyzed the effects of neutrophil degranulation products (polymorphonuclear granulocytes degranulation products, PMN-DGP) on the activation, the adhesion and the migration of three bovine monocyte subsets, as well as their effects on monocyte-macrophage differentiation. Cross-linking of surface CD18 molecules on bovine PMN resulted in the release of primary, secondary and tertiary granules as well as of secretory vesicles. PMN-DGP induced a significant Ca2+-influx in classical (classical monocytes, cM) and intermediate monocytes (intermediate monocytes, intM) but not in non-classical monocytes (non-classical monocytes, ncM). A selective and up-regulated expression induced by PMN-DGP was only seen for CD11a and CD31 on intM. PMN-DGP induced a selective migration of intM in vitro. The presence of PMN-DGP during the differentiation of cM or intM into macrophages resulted in increased expression of membrane CD163 and reduced expression of MHC-II molecules. PMN-DGP-derived macrophages produced more IL-12 and IL-10 and showed enhanced phagocytosis and ROS production capacities. In conclusion, PMN-DGP selectively attract bovine intM and skew the functional maturation of cM and intM

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics.

Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated

MOESM3 of Comparison of the pathogen species-specific immune response in udder derived cell types and their models

Additional file 3. Modulated mRNA concentrations after stimulating pbMEC, pbMFC, RAW 264.7 or MAC-T with E. coli 1303 for various times to illustrate basic and full-scale mRNA expression in these cell types. Values are means from two biological replica experiments, each assayed in duplicate (± SEM) of relative copy numbers; grey underlay, significant induction; red underlay, significant down regulation, fold change > 2, P < 0.05 vs. unstimulated control. Bonferroni’s correction for multiple analyses was applied

MOESM2 of Comparison of the pathogen species-specific immune response in udder derived cell types and their models

Additional file 2. Sequences of the oligonucleotide primers used for real-time PCR quantification. List of the primers used for RT-qPCR, of the pertinent source files and the resulting amplicon sizes

Antibodies used for immunohistochemistry.

<p>Antibodies used for immunohistochemistry.</p

Results of the functional investigation in adolescent WT and SCCx43KO^-/- mice.

<p>Results of the functional investigation in adolescent WT and SCCx43KO^-/- mice.</p

Claudin-3 immunohistochemistry in adult WT and <i>SCCx43KO</i>^<i>-/-</i> mice.

<p>(A) WT tubule showing an immunopositive reaction in the basal part at stage VIII of the seminiferous epithelium (arrows). Scale bar: 50 μm. The positive immunoreaction in the epididymis confirms the binding specificity of the antibody (A, inset). (B) <i>SCCx43KO</i>^<i>-/-</i> tubules with SCO not showing any immunopositive reaction. Scale bar: 50 μm. (C) <i>SCCx43KO</i>^<i>-/-</i> tubules with residual spermatogenesis containing spermatocytes and round spermatids showing a heterogeneous staining pattern with immunopositive and negative reactions in the basal part, apparently independent from the containing GC population. The asterisks indicate two tubules containing spermatocytes. The left one shows an immunopositive reaction for claudin-3 at the basal part of the seminiferous epithelium, the right one is immunonegative. The arrows indicate an immunopositive (right) and an immunonegative (left) reaction in tubules containing round spermatids. Scale bar: 100 μm. (D) <i>SCCx43KO</i>^<i>-/-</i> tubules with qualitative normal spermatogenesis showing the same stage dependent staining pattern as adult WT mice with an immunopositive reaction at stage VIII of the seminiferous epithelium (arrow). Scale bar: 50 μm.</p

Graphic respresentation of the analysed qRT-PCR between adolescent WT and <i>SCCx43KO</i>^<i>-/-</i> mice.

<p>The horizontal line represents the null hypothesis (fold change equal to 1). The small circles denote the fold changes of the mRNA expression in <i>SCCx43KO</i>^<i>-/-</i> mice (day 2 p.p.: 1.05, day 8 p.p.: 1.11; day 10 p.p.: 0.89, day 12 p.p.: 0.94, day 14 p.p.: 1.23, respectively). The vertical lines denote the corresponding 95%-confidence interval. A 95%-confidence interval including the null hypothesis represents a statistically non-significant result.</p

Claudin-5 immunohistochemistry in adult WT and <i>SCCx43KO</i>^<i>-/-</i> mice.

<p>(A) WT tubule showing an immunopositive reaction around spermatocytes in the basal part at stage VIII of the seminiferous epithelium (arrow). Scale bar: 50 μm. (B) <i>SCCx43KO</i>^<i>-/-</i> tubule with SCO not showing any immunopositive reaction. Endothelial cells show an immunopositive reaction (arrow), confirming the binding specificity of the antibody. Scale bar: 50 μm. (C) <i>SCCx43KO</i>^<i>-/-</i> tubules with residual spermatogenesis showing an immunopositive reaction around spermatocytes (arrows). Scale bar: 50 μm. (D) <i>SCCx43KO</i>^<i>-/-</i> tubule with qualitative normal spermatogenesis showing the same staining pattern as adult WT mice (arrows). Scale bar: 50 μm.</p