Jayne Hope

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

ABSTRACT Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells.

The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin‐dependent uptake mechanisms, endocytic pathways involving clathrin‐coated pits, and the mannose receptor. Furthermore, co‐localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established. J. Leukoc. Biol. 66: 50–58; 1999.

Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll-like receptor agonists

Toll‐like receptors (TLR) have been described as partially sharing signalling pathways but showing unique ligand specificity and tissue distribution. Here, the response of bovine macrophages (Mφ) and dendritic cells (DC), both derived from monocytes, was compared by exposing them to the TLR‐specific ligands lipopolysaccharide, poly(I:C)‐double‐stranded RNA, and CpG‐DNA, as well as inactivated Gram‐negative and Gram‐positive bacteria, shown to bind to TLR. The production of NO, superoxide anion, interleukin‐10 (IL‐10), IL‐12 and tumour necrosis factor (TNF) was determined. Compared to monocytes, Mφ expressed more TLR2 and similar levels of TLR4 mRNA transcripts, as analysed by quantitative polymerase chain reaction, whereas DC expressed reduced amounts. Although both DC and Mφ recognized the TLR ligands, dramatic differences were seen in their reaction pattern to them. Both cell types responded with the production of TNF, but DC produced more IL‐12, whereas Mφ produced more IL‐10, regardless of the TLR agonist used. Co‐stimulation with interferon‐γ influenced the amount of cytokine production, but did not alter the cell type‐specific response pattern. Compared to Mφ, DC produced > 10 times less NO upon triggering with TLR ligands. In addition, DC produced superoxide anion to opsonized and non‐opsonized zymosan, but not to phorbol 12‐myristate 13‐acetate, a response pattern confirmed for human Mφ and DC, respectively. Different protein kinase C isoforms and extracellular signal‐regulated kinase patterns were detected in cell lysates of resting and stimulated Mφ and DC. Collectively, our results point to profound differences in pathogen‐derived signal–response coupling occurring commensurate with distinct functions carried out by Mφ or DC.

Caveolae and caveolin in immune cells: distribution and functions

Caveolae are small, cholesterol-rich, hydrophobic membrane domains, characterized by the presence of the protein caveolin and involved in several cellular processes, including clathrin-independent endocytosis, the regulation and transport of cellular cholesterol, and signal transduction. Recently, caveolae have been identified as providing a novel route by which several pathogens are internalized by antigen-presenting cells and as centers for signal transduction. Here, we review the distribution and role of caveolae and caveolin in mammalian immune cells.

NKp46 defines a subset of bovine leukocytes with natural killer cell characteristics

Natural killer (NK) cells have not previously been precisely identified or characterized in cattle or any other ruminant species. We have generated a monoclonal antibody against bovine NKp46, which is expressed exclusively by NK cells in man. NKp46+ cells comprised 1–10% of blood mononuclear cells in cattle, and did not stain with antibodies against CD3, CD4, TCR1, B cell or granulocyte markers. The majority of the NKp46+ cells expressed CD2, and a variable fraction also expressed CD8. The tissue distribution of NKp46+ cells in cattle was compatible with the tissue distribution of NK cells in other species. Bovine NKp46+ cells had typical, large granular lymphocyte morphology, and proliferated vigorously in response to bovine IL‐2 for a limited number of cell divisions. IL‐2‐activated NKp46+ cells killed the bovine kidney cell line MDBK. This cytotoxicity was inhibited by preincubation with antibody against NKp46. In a redirected lysis assay, IL‐2‐activated NKp46+ cells killed the FcγR+ target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is anactivating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.

Development of an ELISA for bovine IL-10

The objective of the study was to develop an assay for bovine IL-10 that could be applied to analyses of immune responses and advance understanding of a variety of diseases of cattle. Recombinant bovine IL-10 (rbo IL-10) was transiently expressed in Cos-7 cells and shown to inhibit the synthesis of IFN gamma by bovine cells stimulated with antigen in vitro. Mice were immunised with a plasmid containing a cDNA insert encoding rbo IL-10 and inoculated with rbo IL-10. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-10 in an ELISA. Some of these mAb neutralised the ability of rbo IL-10 to inhibit IFN gamma synthesis by antigen-stimulated bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-10 present in supernatant of PBMC stimulated with ConA. A luminescent detection method was applied to the ELISA making it more sensitive. Using this method native IL-10 was detected in supernatants of PBMC, diluted blood and undiluted blood from cattle immunised with Mycobacterium bovis BCG or ovalbumin and incubated in vitro with antigen indicating the applicability of the assay to a number of in vitro culture systems.

Existence of CD8α-Like Dendritic Cells with a Conserved Functional Specialization and a Common Molecular Signature in Distant Mammalian Species

The mouse lymphoid organ-resident CD8α+ dendritic cell (DC) subset is specialized in Ag presentation to CD8+ T cells. Recent evidence shows that mouse nonlymphoid tissue CD103+ DCs and human blood DC Ag 3+ DCs share similarities with CD8α+ DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26+ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8α+ and human blood DC Ag 3+ DCs. This allowed the identification of a common set of phenotypic characteristics for CD8α-like DCs in the three mammalian species (i.e., SIRPlo, CADM1hi, CLEC9Ahi, CD205hi, XCR1hi). Compared to CD26− DCs, the sheep CD26+ DCs show 1) potent stimulation of allogeneic naive CD8+ T cells with high selective induction of the Ifnγ and Il22 genes; 2) dominant efficacy in activating specific CD8+ T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8α+-like DCs across mammalian species and identify molecular candidates that could be used for the design of vaccines applying to mammals in general.

Tumor Necrosis Factor Blockers Influence Macrophage Responses to Mycobacterium tuberculosis

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine that mediates inflammation in response to various pathogens, including Mycobacterium tuberculosis, but is also a key factor in the pathogenesis of rheumatoid arthritis and other autoimmune diseases. Three TNF-alpha-suppressing drugs have been approved to treat selected autoimmune diseases; 2 are monoclonal antibodies against TNF-alpha (adalimumab and infliximab), and the other is a soluble TNF receptor/Fc fusion protein (etanercept). TNF blockers have been shown to increase the risk of reactivation of latent tuberculosis, and this risk is higher in patients treated with the monoclonal antibodies. We studied the effects of TNF-alpha blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. All 3 drugs had an inhibitory effect on IFN-gamma-induced phagosome maturation in phorbolmyristate acetate-differentiated human THP-1 cells. Adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages in the presence or absence of IFN-gamma. Treatment of macrophages with TNF-alpha led to increased maturation of phagosomes containing Mycobacterium bovis bacillus Calmette-Guérin or M. tuberculosis H37Rv. These results suggest a role for TNF-alpha in activating phagosome maturation and highlight a mechanism through which TNF-alpha blockade can affect the host response to mycobacteria.

Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis

Vaccination of neonates with Mycobacterium bovis bacillus Calmette–Guérin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2‐week‐old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG‐vaccinated cattle. Levels of interferon gamma (IFN‐γ) detected by enzyme‐linked immunosorbent assay (ELISA) or enzyme‐linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post‐challenge were lower in vaccinated calves. Low IL‐10 responses were evident but IL‐4 was not detected. Responses to ESAT‐6 and/or CFP‐10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT‐6 and CFP‐10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70

ABSTRACT Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-γ) production by NK cells was seen in approximately 30% of noninfected calves in response to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN-γ by NK cells in whole blood in response to ESAT-6 and MPP14 was demonstrated using intracellular staining together with surface labeling for the NK cell-specific receptor, NKp46, or CD3. Furthermore, the depletion of NK cells from peripheral blood mononuclear cells completely abolished the IFN-γ production. The response was mediated through stimulation of adherent cells and was largely independent of contact between adherent cells and the NK cells. Neutralization of interleukin-12 only partly inhibited IFN-γ production, showing that other cytokines were also involved. The demonstration of NK cell-mediated IFN-γ production in young cattle provides an explanation for the nonspecific IFN-γ response frequently encountered in young cattle when using the IFN-γ test in diagnosis of mycobacterial infections.