Late production of CXCL8 in ruminant oro-nasal turbinate cells in response to Chlamydia abortus infection.

Chlamydia abortus is an obligate intracellular bacterium that is an important cause of ovine abortion worldwide. There are reports of abortions in cattle, but these are very rare compared to the reported incidence in sheep. The bacterium is transmitted oro-nasally and can establish a sub-clinical infection until pregnancy, when it can invade the placenta and induce an inflammatory cascade leading to placentitis and abortion. Early host-pathogen interactions could explain differential pathogenesis and subsequent disease outcome in ruminant species. In this study, we assessed the ability of sheep and cattle oro-nasal turbinate cells to sense and respond to C. abortus infection. The cells expressed toll like receptor (TLR) 2, TLR4, nucleotide oligomerization domain (NOD) 1 and NOD-like receptor pyrin domain containing 3 (NLRP3) mRNA. In response to C. abortus infection, both ovine and bovine turbinate cells produce CXCL8 mRNA and protein late in the bacterial developmental cycle, but do not produce IL-1ß or TNF-α. The UV-inactivated bacteria did not elicit a CXCL8 response, suggesting that intracellular multiplication of the bacteria is important for activating the signalling pathways. The production of innate immune cytokines from cattle and sheep turbinate cells in response to C. abortus infection was found to be largely similar.

Lymphoid and myeloid cell populations in the non-pregnant human Fallopian tube and in ectopic pregnancy.

Lymphoid and myeloid cell populations in human endometrium are well-documented and are known to play important roles in providing immune tolerance, controlling trophoblast invasion, and mediating vascular remodeling. Immune cell populations in the Fallopian tube have not been comprehensively studied. The aim of this study was to characterize lymphoid and myeloid cell populations in non-pregnant Fallopian tube and determine whether they are altered in Fallopian tube from women with ectopic pregnancy. Fallopian tube was analyzed by flow cytometry and immunohistochemistry. Populations of CD3+ (CD4+ and CD8+) lymphocytes, LIN1-HLADR+ (CD123+ and CD11c+) dendritic cells, monocytes, neutrophils, and CD56(dim)CD16- natural killer (NK) cells were demonstrated to be present in non-pregnant Fallopian tube. CD123+ dendritic cells were predominant over CD11c+ dendritic cells. Numbers of CD11c+ cells were significantly higher in the progesterone-dominant mid-luteal phase of the menstrual cycle compared with the follicular phase. Numbers of CD45+ leukocytes, CD68+ cells, and CD11c+ cells were higher in Fallopian tube from women with ectopic pregnancy compared with mid-luteal phase Fallopian tube. These data will advance our understanding of normal human Fallopian tube physiology and disorders of Fallopian tube function, such as ectopic pregnancy.

Ectopic lymphoid tissue formation in the lungs of mice infected with Chlamydia pneumoniae is associated with epithelial macrophage inflammatory protein-2/CXCL2 expression.

Infection with Chlamydia pneumoniae (Cp) accounts for around 10% of community acquired bacterial pneumonia and has been associated with other chronic inflammatory conditions. We describe a C57/Bl6 murine model of Cp lung infection characterized by a dose-dependent, resolving neutrophilia followed by lymphocytic infiltration of the lungs. By 21 days post-infection, mice exhibit a T helper type 1 (Th1) polarized serum antibody response with local mucosal antibody secretion and organization of ectopic lymphoid tissue which persisted in the absence of detectable Cp DNA. Macrophage inflammatory protein (MIP)-2/CXCL2, which recruits neutrophils and lymphocytes and is associated with ectopic lymphoid tissue formation, was secreted in the lungs post-infection. In vitro, lung epithelial cells up-regulated MIP-2/CXCL2 in response to both rough lipopolysaccharide (reLPS) and Cp infection. We conclude that Cp infection can have long-term inflammatory effects on tissue that persist after clearance of active infection.

Relative quantitative kinetics of interferon-gamma and interleukin-10 mRNA and protein production by activated ovine peripheral blood mononuclear cells.

Interferon-gamma (IFN-gamma) and interleukin (IL)-10 are cross-regulatory cytokines capable of driving and controlling the adaptive host immune response. The inter-relationship between IFN-gamma and IL-10 expression has not been defined in sheep despite biological evidence suggesting that they perform similar functions to their orthologues described in other species. To address this, we have developed a quantitative (q)PCR method to assess relative levels of IFN-gamma and IL-10 mRNA expression in activated ovine peripheral blood mononuclear cells (PBMC) and compared the kinetics of mRNA expression with amounts of cytokine secreted by the cells over a 96h period. PBMC were collected from sheep immunised with the nominal antigen ovalbumin (Ova) and re-stimulated in vitro with antigen and the T cell mitogen concanavalin A (ConA). The recall response to antigen was characterised by a single peak in IFN-gamma mRNA expression at 48h of culture (13-fold increase over unstimulated cells) and relatively lower expression of IL-10 mRNA (average 2-3-fold increase over the 96h culture period). Antigen-driven IFN-gamma protein concentration was greatest at the end of the culture period (96h) whereas IL-10 protein level was not elevated above that observed in unstimulated cells. The typical response to ConA was greater for both cytokines, with IFN-gamma mRNA expression peaking at 6h of culture (133-fold increase) then declining rapidly whereas IL-10 mRNA expression peaked at 24h (16-fold increase) and declined more gradually. Despite these differences in the relative kinetics of mRNA expression in mitogen-activated PBMC, the typical pattern of protein expression of the two cytokines was similar. Both showed a gradual rise in protein concentration starting from 12h of culture which was still rising at the end of the culture period (96h). These data demonstrate that the kinetics of mRNA expression for IFN-gamma and IL-10 in activated ovine PBMC do not necessarily correlate with detectable protein in culture.

Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF-alpha.

Tumour necrosis factor alpha (TNF-alpha) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-alpha production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-alpha, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-alpha. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-alpha. This ELISA did not detect recombinant ovine (ov) TNF-alpha. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-alpha in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-alpha in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-alpha in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-alpha. These mAbs have generated the capability to study the role of TNF-alpha in host immune protection and disease pathogenesis in ruminants.

Immune response of cows fed polyunsaturated fatty acids under high ambient temperatures.

The aim of the experiment was to determine the effects of 2 different fat supplementations on immune functions of dairy cows under high ambient temperatures. The experiment involved 24 Italian Friesian cows, divided into 3 groups of 8 animals, that were subjected to fat supplementations based on whole flaxseed (FS) or microencapsulated fish oil (FO). At d 0, 45, and 90 of the experiment, lymphocyte response to phytohemagglutinin (PHA) was determined in vivo on each animal by measurement of skin-fold thickness at the site of PHA injection. A humoral response to chicken egg albumin (OVA) was established following a subcutaneous injection with OVA. To assess cows' immune responses, plasma was prepared from experimental blood samples taken at d 0, 15, 30, 45, 60, 75, and 90 of the experiment. Plasma samples were measured for the presence of anti-OVA IgG, IL-1beta, IL-6, and IL-10. Results revealed greater skin-fold thickness in cows fed FS compared with the FO and the control groups, corresponding to higher mean lymphocyte proliferation following in vivo PHA injection. Cows fed FS displayed higher titers of anti-OVA IgG than the control and FO-fed cows. No effects of the diet on IL-1beta or IL-6 were found, whereas IL-10 secretion was lower in FS-fed cows than in control cows. The present study demonstrates that feed supplementation of n-3 polyunsaturated fatty acids can enhance immune responses of dairy cows exposed to high ambient temperatures.

Antigen-specific peripheral immune responses are unaltered during normal pregnancy in sheep.

A shift in the balance of T(Helper) (T(H))1/T(H)2 cytokine production by maternal peripheral blood leukocytes is regarded as a common important feature of successful mammalian pregnancy. Although the phenomenon has been studied extensively in animals with invasive hemochorial placentae, the paradigm has not been studied in detail in species with less-invasive placentae, such as sheep that have a synepitheliochorial placenta. Sixteen sheep were immunised with the nominal antigen chicken egg albumin (Ova) and antigen-specific humoral and cellular responses were established in all sheep. The 16 sheep were synchronised, 11 were mated and successfully conceived, the remaining 5 served as non-pregnant controls. Peripheral blood mononuclear cells (PBMC) were isolated approximately every 2 weeks and restimulated in vitro with either Ova or the T cell mitogen concanavalin A (ConA), and cell proliferation and cytokine production measured. There were no detectable differences in antigen-driven PBMC proliferation, interferon-gamma (IFN-gamma), interleukin (IL)-4 or IL-10 production between pregnant and non-pregnant sheep. Also, there were no appreciable differences in ConA-induced IFN-gamma, IL-4 or IL-10 between the groups. These data suggest that a shift in T(H)1/T(H)2 cytokine production does not occur in pregnant sheep and indicate that further comparative reproductive immunology studies on species with non-invasive placentation will be informative of materno-fetal interactions and immune regulation during pregnancy.

Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Immunopathology of Chlamydophila abortus infection in sheep and mice.

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.

Variability in cytokine production and cell proliferation by mitogen-activated ovine peripheral blood mononuclear cells: modulation by interleukin (IL)-10 and IL-12.

T-cell reactivity is typically measured by cell proliferation and/or production of cytokines in response to antigenic/mitogenic stimulation. The choice of assays is more limited in ruminants than rodents, and complicated by the variability inherent in outbred populations. We have measured proliferation and production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC) from 24 sheep, and compared the responses between sheep, within sheep over several sample points, and also drawn comparisons between the two assays. PBMC derived from different sheep varied by as much as ten-fold in both proliferation and IFN-gamma production, though not necessarily at the same sample time. Thus, there was a poor correlation between the two assays and also considerable variation in the responses from the same animal at different time points. Both parameters could be modulated by exogenous recombinant ovine interleukin (IL)-10 and IL-12, but we were unable to correlate IFN-gamma production with endogenous cytokine production in the assays. These data highlight the importance of assay selection for the measurement of immune responsiveness and also demonstrate the variation that can be expected between sheep and over time.

Ovine chlamydial abortion: characterization of the inflammatory immune response in placental tissues.

Ovine chlamydial abortion is a serious cause of fetal mortality in several sheep-rearing countries. The causal agent, Chlamydophila abortus (Chlamydia psittaci), does not generally induce clinical signs in the ewe other than abortion; this is associated with macroscopically visible damage in the placenta, which may be inflamed and thickened. To investigate the nature of the placental inflammation, seven pregnant sheep were inoculated subcutaneously at 70 days' gestation with C. abortus (strain S 26/3). A further five pregnant sheep received control inoculum by the same route at the same stage of pregnancy. Three of the infected ewes produced stillborn lambs and four produced live lambs. Lesions characteristic of chlamydial infection were present in all placentas except for two from one ewe that gave birth to twins. Histopathological examination of placental tissues from aborted fetuses showed a mixed inflammatory cell infiltrate with vasculitis and thrombosis in the mesenchyme of the intercotyledonary membranes. Cells expressing the macrophage-associated molecule CD 14 were found to be numerous, as were cells expressing major histocompatibility complex class II (MHC II) molecules. Many cells expressing messenger RNA (mRNA) encoding for tumour necrosis factor-alpha (TNF-alpha) were demonstrated, but few cells expressing interferon gamma mRNA and none expressing interleukin-4 mRNA were detected. The fetal immune response included small numbers of CD4+ and CD8+ cells, gamma delta T cells and B cells. It is concluded that abortion is the result of several factors, including destruction of tissue by C. abortus, vascular thrombosis, and an inflammatory response by the fetus. Production of TNF-alpha by fetal macrophages expressing MHC II molecules may be of considerable significance in the pathogenesis of abortion.

Development of detection methods for ruminant interleukin (IL)-12.

Recombinant bovine IL-12 (rbo IL-12) was transiently expressed in COS-7 cells and shown to upregulate the synthesis of IFNgamma by bovine cells stimulated with a suboptimal concentration of mitogen in vitro. Mice were immunised with a plasmid encoding rbo IL-12 and boosted with rbo IL-12 and a number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-12 in an ELISA. Some of these mAb neutralised the ability of rbo IL-12 to induce IFNgamma synthesis by bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-12 by ELISA and a luminometric detection method was applied to the ELISA making it more sensitive. Using this method native bovine IL-12 was detected in supernatants of dendritic cells (DC) cultured in vitro with a synthetic lipopeptide known to stimulate secretion of IL-12 by human DC. The ELISA was also able to detect recombinant ovine IL-12 and, less effectively, recombinant human IL-12. In contrast, bovine IL-12 was not detected by a commercial human IL-12 ELISA kit. Intracytoplasmic IL-12 was detected in bovine DC using the antibodies described herein. The ability to detect ruminant IL-12 by three methods: ELISA, bioassay with neutralising mAb and cytoplasmic staining, will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Immune regulation during pregnancy and host-pathogen interactions in infectious abortion.

The immunological mechanisms that govern the success of pregnancy in outbred mammals are complex. During placental formation the invasion of fetal cells into maternal tissue must be controlled to prevent damage to the mother. Equally, maternal recognition of pregnancy must be such that allorejection of the fetus does not occur. Despite the complexity of this phenomenon, it is clear that cytokines play a crucial role at the maternofetal interface and in the periphery to ensure that pregnancy proceeds successfully. Inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) can exert detrimental effects in the placenta and tend to be present at low concentrations, whereas the regulatory cytokines interleukin (IL)-10 and tranforming growth factor-beta (TGF-beta) are beneficial and tend to predominate. This means that infection with pathogens that target the placenta and that elicit inflammatory responses may cause abortion by giving rise to a detrimental combination of cytokines that causes damage but does not control the disease. Infectious abortion is discussed in the context of the modulation of host immune responses during pregnancy, taking into account the different placental structures present in human beings, rodents and ruminants.

A role for tryptophan in immune control of chlamydial abortion in sheep.

Tryptophan (Trp) catabolism appears to be an important mechanism for regulation of inflammatory responses, resulting in T-cell tolerance and survival of semi-allogeneic concepti during pregnancy. Trp catabolism can be induced by IFN-gamma, and is therefore an important host defence mechanism against intracellular pathogens. Chlamydophila abortus is a bacterial pathogen that can cause persistent infection in non-pregnant sheep, but invades the placenta and causes abortion in late pregnancy. IFN-gamma was found to control the growth of Chlamydophila abortus in ovine cells in a highly dose-dependent manner. Addition of 200U/ml IFN-gamma eradicated all traces of infection from the cultures, whereas concentrations less than 50U/ml failed to control the growth of the organism, resulting in cell lysis. However, concentrations in the range of 50-100U/ml were found to restrict growth to an extent that a persistent infection was established, allowing survival of the organism in tissue culture for several months. Removal of IFN-gamma resulted in the re-appearance of infectious organisms. Addition of exogenous Trp to the cells treated with 50-100U/ml IFN-gamma prevented the establishment of persistence. These effects in tissue culture are analogous to the persistent infection observed in pregnant sheep prior to abortion. These data suggest that control of C. abortus growth in the periphery is linked to the balance of pro-inflammatory cytokine production and availability of Trp during pregnancy.

Cytokine release by ovine macrophages following infection with Chlamydia psittaci.

Chlamydia psittaci is an obligate intracellular pathogen that causes abortion in both sheep and humans. The disease in sheep (but not humans) is characterized by a long-term persistent phase that appears to be under the control of interferon-gamma. However, nothing is known about cytokine induction that precedes the persistent phase in sheep. Primary alveolar lavage cells recovered from normal adult sheep were used to study cytokine production in the first 72 h of infection with C. psittaci. These cells were phenotypically characteristic of macrophages, being adherent, phagocytic, CD14+ and staining positive for non-specific esterase. In vitro infection of the macrophages with C. psittaci resulted in the release of IL-1beta, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as measured by ovine-specific ELISAs. Heat-treated chlamydiae (1 h at 65 degrees C) did not induce the release of IL-1beta, but the release of IL-8 was similar to that induced by untreated organisms. The cells from different sheep varied most notably in their patterns of GM-CSF release in response to heat-treated and untreated organisms.

Antibody and cytokine responses in efferent lymph following vaccination with different adjuvants.

The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2 titres were higher and maintained for longer periods of time in lymph from sheep vaccinated with IFA and lower and shorter lived in animals which received the Al(OH)3 based vaccine. Interferon-gamma was present within one day in efferent lymph from all sheep which received the Quil A formulation and in only one of the three sheep that received the IFA formulation. GM-CSF was only detected in lymph from sheep vaccinated with the IFA formulation. IL-8 was present in lymph prior to vaccination and only animals which received the Quil A formulation had increased levels of IL-8 after vaccination. Neither of the inflammatory cytokines IL-1 beta and TNF alpha were detected in efferent lymph from any animals in this study. This paper highlights the potential of the lymphatic cannulation model for investigations of the in vivo action of adjuvants.

Cytokines and the protective host immune response to Chlamydia psittaci.

The immunobiology of enzootic abortion of ewes (EAE) is incompletely understood. The causative agent is Chlamydia psittaci, which infects many ruminant species and has zoonotic potential. The organism can survive in the ovine host for many months without causing clinical symptoms but does not generate a sterile immunity during this time. It has been postulated that the organism persists in the host entering at a latent phase, possibly mediated by host cytokine production. The effects of cytokines on chlamydial multiplication vary between host species, between different cell types within those species and also vary between chlamydial species and strains. The multiplication of the EAE strain of C. psittaci in ovine ST-6 cells can be restricted by interferon-gamma (IFN-gamma) but not with comparable concentrations of IFN-alpha. Altering the nutrient composition of the cultures by addition of tryptophan partially reverses the antichlamydial effects of the IFN-gamma. This offers a potential mechanism by which C. psittaci can persist in sheep. The implications of these observations for the pathogenesis of EAE are discussed.

Protective immune responses to Theileria annulata of relevance to vaccine development.

A series of projects on Theileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages of T. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.

Interferon-gamma mediates long-term persistent Chlamydia psittaci infection in vitro.

The long-term anti-chlamydial effects of recombinant ovine interferon gamma (rOvIFN-gamma) were studied in ovine ST-6 fibroblasts infected with the S26/3 strain of Chlamydia psittaci. Chlamydial multiplication was assessed by enzyme-linked immunosorbent assay analysis of supernate lipopolysaccharide, titration of inclusion-forming units in culture supernates, and enumeration of inclusion bodies in cultured cells at 7-day intervals. Concentrations of 250 and 1000 U/ml of rOvIFN-gamma resulted in a microbistatic inhibition of C. psittaci growth, which appeared to become microbicidal when rOvIFN-gamma was maintained in the cultures for 14 days or more. There were no signs of C. psittaci multiplication when cultures were maintained in 25 or 100 U/ml of rOvIFN-gamma. However, subsequent removal of rOvIFN-gamma from these cultures resulted in a re-emergence of viable, infectious chlamydiae, which eventually killed all the fibroblasts. This re-emergence was more rapid in cultures initially treated with 25 U/ml of rOvIFN-gamma than in those treated with 100 U/ml.