Joanne Cummerson

Dynamic resolution of acrosomal exocytosis in human sperm.

An essential step in mammalian fertilisation is the sperm acrosome reaction (AR) – exocytosis of a single large vesicle (the acrosome) that surrounds the nucleus at the apical sperm head. The acrosomal and plasma membranes fuse, resulting in both the release of factors that might facilitate penetration of the zona pellucida (which invests the egg) and the externalisation of membrane components required for gamete fusion. Exocytosis in somatic cells is a rapid process – typically complete within milliseconds – yet acrosomal enzymes are required throughout zona penetration – a period of minutes. Here, we present the first studies of this crucial and complex event occurring in real-time in individual live sperm using time-lapse fluorescence microscopy. Simultaneous imaging of separate probes for acrosomal content and inner acrosomal membrane show that rapid membrane fusion, initiated at the cell apex, is followed by exceptionally slow dispersal of acrosomal content (up to 12 minutes). Cells that lose their acrosome prematurely (spontaneous AR), compromising their ability to penetrate the egg vestments, are those that are already subject to a loss of motility and viability. Cells undergoing stimulus-induced AR (progesterone or A23187) remain viable, with a proportion remaining motile (progesterone). These findings suggest that the AR is a highly adapted form of exocytosis.

Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non‐lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4+ cells expressed significantly less CD46 than CD8+ cells (P < 0·05) while the reverse was observed for CD55 (P < 0·02). CD56+ cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0·02). CD45RO+ cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO– cells (P < 0·02); CD25+ cells also expressed significantly less CD55 than CD25– cells (P < 0·002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up‐regulated on all leucocyte subsets with the exception of CD56+ cells. Both CD55 and CD59 were also markedly up‐regulated on monocytes, and CD55 expression was greater on CD8+ than CD4+ cells following activation (P < 0·02). Lipopolysaccharide treatment did not significantly alter B‐cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up‐regulated on monocytes (P < 0·02). These observations that CReg proteins are up‐regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

The complement regulatory proteins CD55 (decay accelerating factor) and CD59 are expressed on the inner acrosomal membrane of human spermatozoa as well as CD46 (membrane cofactor protein)

The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation‐competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)‐anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM‐associated CD55 was localised within GM1‐containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI‐PLC, demonstrating them to be GPI‐anchored. Analysis of acrosome‐reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody‐induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site.

Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host.

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3s influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.

Persistent viral infection in humans can drive high frequency low-affinity T-cell expansions

CD8 T cells that recognize cytomegalovirus (CMV) ‐encoded peptides can be readily detected by staining with human leucocyte antigen (HLA) –peptide tetramers. These cells are invariably highly differentiated effector memory cells with high avidity T‐cell receptors (TCR). In this report we demonstrate an HLA‐A*0201 restricted CMV‐specific CD8 T‐cell response (designated YVL) that represents several percent of the CD8 T‐cell subset, yet fails to bind tetrameric major histocompatibility complex (MHC) ligands. However, these tetramer‐negative cells are both phenotypically and functionally similar to other CMV‐specific CD8 T cells. YVL peptide‐specific CD8 T‐cell clones were generated and found to be of high avidity in both cytotoxicity and interferon‐γ (IFN‐γ) assays, and comparable with other CMV peptide‐specific CD8 T‐cell clones. However, under conditions of CD8 blockade, the response was almost nullified even at very high ligand concentrations. This was also the case in IFN‐γ experiments using peripheral blood mononuclear cells stimulated with peptide ex vivo. In contrast, all other CMV specificities (tetramer‐positive) displayed minimal or only partial CD8 dependence. This suggests that YVL‐specific responses depict a low‐affinity TCR–MHC–peptide interaction, that is compensated by substantial CD8 involvement for functional purposes, yet cannot engage multivalent soluble ligands for ex vivo analysis. It is interesting that such a phenomenon is apparent in the face of a persistent virus infection such as CMV, where the responding cells represent an immunodominant response in that individual and may present a highly differentiated effector phenotype.

Neutrophil TLR4 expression is reduced in the airways of infants with severe bronchiolitis

Background: In respiratory syncytial virus (RSV) bronchiolitis, neutrophils account for >80% of cells recovered from the airways in bronchoalveolar lavage (BAL) fluid. This study investigated neutrophil activation and Toll-like receptor (TLR) expression in the blood and lungs of infants with severe RSV bronchiolitis. Methods: BAL fluid and (blood) samples were collected from 24 (16) preterm and 23 (15) term infants ventilated with RSV bronchiolitis, and 12 (8) control infants. Protein levels and mRNA expression of CD11b, myeloperoxidase (MPO) and TLRs 2, 4, 7, 8 and 9 were measured in neutrophils. Results: Blood neutrophils had more CD11b in preterm and term infants with RSV bronchiolitis than control infants (p<0.025) but similar amounts of MPO. BAL fluid neutrophils from infants with RSV bronchiolitis had greater amounts of CD11b and MPO than blood neutrophils and BAL fluid neutrophils from controls (p<0.01). Blood neutrophils from term infants with RSV bronchiolitis had less total TLR4 protein than preterm infants with RSV bronchiolitis (p = 0.005), and both had less than controls (p<0.04). Total TLR4 for each group was greater in BAL fluid neutrophils than in blood neutrophils. Blood neutrophils from preterm infants with RSV bronchiolitis had greater TLR4 mRNA expression than term infants with RSV bronchiolitis (p = 0.005) who had similar expression to controls (p = 0.625). Conclusions: In infants with severe RSV bronchiolitis, neutrophil activation starts in the blood and progresses as they are recruited into the airways. Total neutrophil TLR4 remains low in both compartments. TLR4 mRNA expression is unimpaired. This suggests that neutrophil TLR4 expression is deficient in these infants, which may explain why they develop severe RSV bronchiolitis.