Isoflavonoid compounds from red clover (Trifolium pratense) protect from inflammation and immune suppression induced by UV radiation.

Isoflavones derived from many edible plants have been reported to possess significant antioxidant, estrogenic and tyrosine kinase inhibitory activity. Genistein has been found previously to provide protection from oxidative damage induced by UV radiation both in vitro and following dietary administration. We have therefore examined the potential of a number of isoflavones from red clover (Trifolium pratense) and some metabolically related compounds to offer protection from UV irradiation in hairless mice by topical application after UV exposure. We show that whereas the primary isoflavones, daidzein, biochanin A and formononetin, were inactive, 20 microM lotions of genistein and the metabolites equol, isoequol and the related derivative dehydroequol had powerful potential to reduce the inflammatory edema reaction and the suppression of contact hypersensitivity induced by moderate doses of solar-simulated UV radiation. For equol the protection was concentration dependent and 5 microM equol markedly reduced the UV-induced inflammation but abrogated the UV-induced immunosuppression. Equol protected similarly from immunosuppression induced by the putative epidermal mediator, cis-urocanic acid (UCA), indicating a potential mechanism of action involving inactivation of this UV-photoproduct. Since immunosuppression induced by both UV radiation and by cis-UCA appears to be an oxidant-dependent response our observations support the actions of these topically applied isoflavones and their metabolites as antioxidants. They also indicate that lotions containing equol, unlike topical UV sunscreens, more readily protect the immune system from photosuppression than from the inflammation of the sunburn reaction, even when applied after exposure, and thus such compounds may have a future role as sun-protective cosmetic ingredients.

Effects of exogenous interleukin-6 during Pseudomonas aeruginosa corneal infection.

Lack of interleukin-6 (IL-6) during Pseudomonas aeruginosa corneal infection leads to more severe disease with changes in neutrophil recruitment. Exogenous IL-6 leads to increased efficiency of neutrophil recruitment and reduced bacterial loads in corneal infection in both IL-6 gene knockout and wild-type mice. This may be mediated by IL-6 increasing the production of corneal macrophage inflammatory protein 2 and intercellular cell adhesion molecule 1. We conclude that effective recruitment of neutrophils into the cornea is dependent on the production of IL-6 and that early augmentation of IL-6 may be protective in corneal infection.

Differential interleukin-6 mRNA expression in Nippostrongylus brasiliensis infection of susceptible and resistant strains of mice.

Intestinal parasitic infection is still a major problem in humans and animals, yet host immunity against gut parasitic infection remains partially understood. Eosinophilia and mastocytosis are features of such infection that have been shown to be genetically controlled. The expression of IL-6 is detected in eosinophils, mast cells and neutrophils and may be responsible for the regulation of leucocytes at infective sites. The relationships between IL-6 expression, eosinophilia, mastocytosis and host immunity remain unclear. In the present report, a close correlation between IL-6 mRNA+ cells, eosinophilia, mastocytosis and worm expulsion is demonstrated, which may indicate a role for IL-6 in regulation of host immunity against intestinal parasite infection.

Interleukin-6 expression in gut of parasite challenged sheep.

Following challenge with Trichosirongylus colubrifonizis, increased numbers of T-cells and immunoglobulin responses are seen in the intestine of sheep immunised by repeated infection with live worms. IL-6 mRNA expression in the small intestine from T. colubriformis-immunised and naive sheep was determined by in situ hybridisation, whereas CD4(+), IgA(+), IgG(+) cells in the gut were evaluated by immunohistochemistry. There was constitutive expression of IL-6 mRNA by cells in the naive gut, and the number of these cells was increased by parasite challenge. There were corresponding increases in numbers of CD4(+) and TCR gamma/delta(+) T-cells and IgG(+) B-cells. Our data are consistent with a role for IL-6, perhaps produced by CD4(+) and/or TCR gamma/delta(+) T-cells or B-cells, in B-cell terminal differentiation. Infiltration of B-cells, particularly IgG(+) B-cells, may reflect parasite immunity in the host.

Investigation of the site of precursors for IgA-producing cells in the chicken intestine.

Bursectomized chicks received lymphocyte single cell suspensions harvested from the bursa of Fabricius (BF), ileal lymphoid aggregate (ILA), caecal tonsils (CT), spleen and peripheral blood. Four days after cell transfer, repopulation of the duodenal and CT lamina propria in age-matched recipient bursectomized chickens with IgA-secreting plasma cells was determined. The results indicate the highest level of reconstitution with cells derived from BF, but substantial numbers of IgA-secreting plasma cells were also observed in a number of birds that received lymphocytes originating from the ILA and CT.

In vivo T-cell subset depletion suggests that CD4+ T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep.

In vivo lymphocyte subset depletion offers a unique opportunity to study the roles of different cellular components of the immune system of sheep during infection with orf virus. Lambs were depleted of specific lymphocyte subsets by the intravenous administration of monoclonal antibodies against ovine lymphocyte surface markers and then challenged with orf virus. The skin lesions that developed were scored visually as to their severity. Blood samples were collected to monitor the lymphocyte depletions and to measure orf-virus-specific antibody levels. Skin biopsies were collected from the lesion site and studied to determine the course of the infection and the presence of various cell types and orf virus. All the sheep developed orf virus lesions after infection. All three of the CD4-depleted lambs were unable to clear virus from their skin and did not have an antibody response to the virus. Virus was also detected in the skin of one each of the three CD8-depleted, WC1-depleted and control sheep on the final day of the trial. CD8(+) lymphocytes did not appear to be essential for viral clearance later in the infection. Depletion of the majority of gammadelta(+) T-cells did not affect the outcome of orf virus infection. In sheep with high orf-virus-specific antibody titres at the time of infection, orf lesions healed faster than lesions in sheep with low antibody levels, and this occurred regardless of the lymphocyte depletion status of the animals. This study suggests that the presence of CD4(+) T-cells and orf-virus-specific antibodies are important for the control of viral replication in the skin of infected sheep.

Immunity, vaccination and the avian intestinal tract.

Defence of the intestinal mucosal surface from enteric pathogens is initially mediated by secretory IgA (SIgA). As oral immunization of non-replicating antigen induces minimal SIgA antibody titers, novel immunization strategies which selectively induce mucosal immune responses in mammals are now being assessed in chickens. The strategies reviewed include the route of antigen delivery, the incorporation of antigenic components in delivery vehicles, the inclusion of immunomodulators in the vaccine formula or in the diet, and manipulation of intestinal microflora. The differences in anatomical organization and immunological mechanisms between birds and mammals must be considered when manipulating avian intestinal immunity with the latest immunotechnologies developed for mammals. Our knowledge of the function and functioning of the avian mucosal system is discussed. Progress in our understanding of this system, the location of precursor IgA B cells and antigen sampling by these sites is not as advanced as knowledge of the mammalian system, highlighting the need for ongoing research into the avian application of novel vaccination strategies.

Induction of T helper 1- and T helper 2-type immune responses during Haemonchus contortus infection in sheep.

The production of cytokines by lymphoid cells, isolated from non-infected and Haemonchus contortus-infected lambs, was investigated. Particular attention was paid to differences in T helper 1- (Th1) and Th2-type immune profiles between genetically resistant and random-bred animal groups. Non-infected resistant and random-bred lambs produced equivalent levels of interferon-gamma (IFN-gamma) and interleukin-5 (IL-5), from isolated abomasal lymph node cells (ALN), mesenteric lymph node cells (MLN) and spleen cells (SC), in response to in vitro stimulation with T-cell mitogen (concanavalin A) or larval parasite antigen. ALN and MLN cells derived from infected resistant and random-bred lambs produced relatively lower levels of IFN-gamma, following in vitro stimulation with parasite antigen, when compared with their uninfected counterparts. In contrast, infected lambs of both groups showed enhanced mitogen- and antigen-stimulated production of IL-5, in comparison with uninfected controls, at days 5 and 28 postinfection (p.i.). Mitogen- and antigen-stimulated IL-5 responses were higher among resistant lambs compared with random-bred lambs, with the highest overall production of IL-5 by parasite antigen-stimulated ALN and MLN cells. Among day 28 p.i. lambs, levels of cell culture-derived parasite-specific immunoglobulin G1 (IgG1) and IgE antibodies were higher in resistant lambs than in random-bred lambs, following in vitro stimulation of SC or ALN cells with parasite antigen. Finally, after 28 days p.i., histological examination of abomasal tissue revealed higher densities of mast cells and eosinophils in the mucosa of resistant lambs than in random-bred lambs. Taken together, these data support the notion of a strong Th2-type immune response to Haemonchus infection in genetically resistant sheep, and support the claim for a Th1/Th2 dichotomy in ruminants.

Interferon-gamma plays a critical role in intestinal immunity against Salmonella typhimurium infection.

Salmonella bacteria are a major cause of food-borne infectious diarrhoea and there is great interest in understanding the pathogenesis of Salmonella infection and in vaccine development. Potential vaccines include the aromatic mutants of S. typhimurium. Such non-lethal Aro mutants have also been useful for studying Salmonella infections in mouse models. Studies of systemic infection, using these Aro mutants, in both normal and cytokine gene knockout mice, indicate that interferon-gamma (IFN-gamma) plays a key role in the resolution of Salmonella infection. The present studies have investigated the outcome of oral infection in mice with attenuated Salmonella because this infection route mimics natural infection in humans. In IFN-gamma gene knockout (IFN-gamma-/-) mice, intestinal immunity was impaired and oral challenge resulted in disseminated septicaemia 2 weeks later. No dissemination of infection was seen in wild-type mice. In wild-type mice, both CD4 and CD8 cell numbers increased in the gut following Salmonella challenge, together with increased expression of major histocompatibility complex (MHC) II and vascular cell adhesion molecule-1 (VCAM-1). No such changes were seen in IFNgamma-/- mice. Following oral challenge, antilipopolysaccharide (LPS) and antiphosphoryl choline antibodies increased by more than 100-fold in both serum and faecal pellet extracts of IFNgamma-/- mice compared with wild-type mice. Our data show that IFN-gamma production is essential for resolution of enteric Salmonella infection and that antibody has little effect on this process.

Ontogeny of IgA(+) cells in lamina propria: effects of sympathectomy.

The effect of chemical sympathectomy on the ontogeny of the IgA(+) cells in the intestinal LP was examined in weanling rats. Ablation of the peripheral sympathetic nerve terminals using 6-hydroxydopamine (6-OHDA) on days 14 and 17 was associated with an increase in the number of IgA(+) and IgM(+) cells in the intestinal LP at 28, 30 and 35 days of age. Despite the precocious development of Ig-containing cells in the gut, the specific intestinal immune response to ovalbumin (OVA), induced by IP priming with OVA at 30 days of age and boosting ID 14 days later, was not altered by 6-OHDA treatment, with no difference observed in the numbers of total AOCC or IgA(+)/AOCC in the LP of treated, compared to control animals. The results presented in this study suggest that sympathetic innervation is an influential factor in the ontogeny of IgA(+) cells populating the intestinal lamina propria, although no functional significance in terms of the specific local response to a new antigen was detected using the immunisation model described here.

KC production in the cornea in response to Pseudomonas aeruginosa challenge.

Pseudomonas aeruginosa can cause ulcerative bacterial keratitis. A feature of keratitis is the rapid infiltration of the avascular corneal stroma by neutrophils. KC is a potent neutrophil chemokine. The present study used a mouse model of ocular infection to assess the relationship between KC and inflammation in the cornea in response to challenge with a strain of P. aeruginosa causing keratitis. Low levels of KC mRNA and protein were detected by in situ hybridization and ELISA, respectively, in unchallenged corneas. Dramatically increased numbers of KC mRNA+ cells were present in P. aeruginosa strain 6294-challenged corneas. Expression of KC mRNA was found to be up-regulated in the corneal epithelium in response to wounding alone. The KC mRNA+ cells were located in the epithelium and corresponding to infiltrating neutrophils cells in the stroma. Quantification of KC protein at different time points showed peak levels at 8 h of bacterial challenge. These results suggest that KC may be involved with the regulation of leucocyte infiltration early during bacterial keratitis.

Analysis of the mucosal microenvironment: factors determining successful responses to mucosal vaccines.

The predominance of IgA antibodies in mucosal sites reflects a combination of high rate IgA isotype switching among precursor cells in induction sites, their selective localisation in mucosal effector tissues and vigorous proliferation of these cells after extravasation. Each of these steps leading to IgA expression at the mucosa is under cytokine control. This paper will address the role of cytokines in induction and expression of IgA responses, the contribution of various precursor cell subsets and their differential responses to cytokine signals and strategies for manipulating cytokine expression. With respect to IgA antibody production in the gut whereas IL-4 and TGF-beta have been implicated in isotype switching of precursor cells to IgA commitment, their subsequent localisation, proliferation and effector activity expression is dependent on IL-5 and IL-6 expression locally. Most IgA plasma cells in the intestine derive from cells of the B2 lineage in the Peyer's patch, but a subpopulation of cells derived from the peritoneal cavity (B1 cells) also contribute to the IgA plasma cell population in the intestinal lamina propria. Whereas IgA+ cells of the B2 lineage are IL-6 dependent but IL-5 independent, B1-derived IgA+ cells are IL-5 dependent and IL-6 independent. On the other hand, cell mediated immune responses in the gut are highly dependent on IFN-gamma production by both Th1 CD4 cells and CD8 cells and in enteric Salmonella infection IFN-gamma production is essential but antibody has little effect on this process.Therapeutic interventions based on the information emerging from these studies will lead to improved vaccination responses and correction of immunodeficiencies especially in young animals.

The effect of molecular weight and beta-1,6-linkages on priming of macrophage function in mice by (1,3)-beta-D-glucan.

1,3-beta-D-glucans (glucans) are structural elements in the cell walls of yeast and fungi with immunomodulatory properties, mediated through their ability to activate macrophages. This study assessed the activation of cells of the peritoneal cavity between 3 and 90 days after i.p. injection of particulate yeast glucan differing in molecular weight (MW) and degree of (1,6)-linkages. Female QS mice, 7-9 weeks of age, were injected, i.p., with varying doses of low (< 5 x 10(5)), medium (1-2 x 10(6)) or high (> 3 x 10(6)) MW glucans, all with low (< 5%) beta-(1,6)-linkages, or high MW (> 3 x 10(6)) glucan with high 1,6-linkages (> 20%). All glucans induced a transient increase in the proportion of neutrophils and eosinophils and a reduction in mast cell numbers in the peritoneal cavity. Peritoneal macrophages showed an altered morphology, increased intracellular acid phosphatase, increased LPS-stimulated NO production and increased PMA-stimulated superoxide production. There were no significant changes in serum lysozyme levels. Most macrophage activities returned to control levels by 28 days post injection of 1, 3-beta-D-glucan. There was a trend for higher MW or (1,6)-linked, (1, 3)-beta-D-glucans to be more stimulatory. It was concluded that particulate yeast (1,3)-beta-D-glucan is an effective stimulator of immune function, the efficiency of which may be influenced by the MW and degree of (1,6)-linkages.

Sympathectomy abrogates immunodeficiency associated with protein-energy malnutrition.

The role of the sympathetic nervous system in the immune deficiency developed in protein-energy malnutrition (PEM) was investigated by assessing the effects of sympathectomy on the intestinal immune response of rats subject to prenatal or postnatal malnutrition. Chemical sympathectomy increased the number of IgA+ cells migrating into the intestinal lamina propria of control animals, but this effect was abrogated in rats malnourished during their perinatal stage. The method by which perinatal malnutrition was achieved influenced the magnitude of the effect on serum IgA levels with malnutrition during lactation having a more pronounced depressive effect on IgA than malnutrition during gestation. In experiments in which animals were intestinally immunised with ovalbumin (OVA) the mucosal immune response was reduced in non-sympathectomised malnourished (MN) animals and a lower level of anti-OVA IgA was detected in serum. However, in sympathectomised animals, there was no difference between MN animals and controls in the intestinal and humoral immune responses. The preliminary evidence presented in this paper strongly supports a role for the noradrenergic neurotransmitter system in the immunodeficiency developed during PEM.

Characterization of a bioassay for detection of recombinant and native ovine interleukin-5.

In order to analyse Th2-type immune responses in sheep by the assay of interleukin (IL)-5 in biological fluids, the ovine IL-5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC-OV-IL-5 from the supernatant fluid. The ovine IL-5 was biologically active in a bioassay using IL-5-dependent Baf cells, which have been used previously to specifically detect human IL-5. The specificity of Baf cells for ovine IL-5 was examined by two methods. First, Baf cells only proliferated in response to BAC-OV-IL-5 and did not respond to addition of recombinant ovine cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1beta, IL-2, IL-3, IL-6, IL-8, stem cell factor (SCF) or IFN-gamma at doses from 0.01 to 1 microg/well. Second, the rat monoclonal antibody to murine IL-5, TRFK-5, neutralized murine, but not ovine, IL-5. However, rabbit antisera to BAC-OV-IL-5 neutralized murine and ovine recombinant IL-5 and abolished responses of Baf cells to IL-5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 microL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL-5 also extends to ovine IL-5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.

Expression of interleukin-6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa causing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains of P. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model with P. aeruginosa 6294, 6206, or Paer1 (2 x 10(6) CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates of P. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains of P. aeruginosa correlated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.

TNF-alpha production in the cornea in response to Pseudomonas aeruginosa challenge.

Pseudomonas aeruginosa can cause ulcerative bacterial keratitis or contact lens-induced acute red eye (CLARE) in humans. The present study used a mouse model of ocular infection and inflammation to examine the relationship between TNF-alpha and inflammation in the cornea in response to challenge with either a strain of P. aeruginosa causing keratitis or a CLARE strain. Constitutive TNF-alpha mRNA was detected in the epithelium, mainly towards the periphery. After infection with the keratitis-inducing strain (6294), TNF-alpha expression was elevated four-fold by 24 h post-challenge. No detectable induction of TNF-alpha mRNA was seen with CLARE strain (Paer1) challenge at any time point. The TNF-alpha protein production detected by ELISA showed a corresponding pattern to the mRNA expression, which also correlated with pathological changes. These results suggest that invasive strains of P. aeruginosa create greater pathological changes as a result of elevated TNF-alpha production, which contributes to inflammation during keratitis in vivo.

B1 B cell numbers and antibodies against phosphorylcholine and LPS are increased in IL-6 gene knockout mice.

Peritoneal cavity cells were isolated from IL6-gene knockout (IL6(-/-)) and wild-type mice and stained for expression of IgM, CD5, and CD23. B1 cell (IgM(+)/CD23(-), CD5(+)/IgM(+)) numbers were increased twofold in IL6(-/-) mice compared to normals while IgM(+)/CD23(+) (B2) cell numbers were reduced threefold. Intestinal antibody levels were also determined for both total immunoglobulin and phosphorylcholine (PC)-specific and LPS-specific antibody following oral challenge with attenuated Salmonella typhimurium. Total immunoglobulin levels (IgM, IgG, and IgA) were reduced 60-80% in intestinal secretions of IL6(-/-) mice compared to wild-type controls; however, PC-specific antibody was significantly higher in IL6(-/-) mice. Anti-LPS antibodies were also three- to sevenfold higher in IL6(-/-) mice compared to controls following Salmonella challenge. These data suggest that in IL6(-/-) mice the development of mucosal B2 cells is impaired but that intestinal B1 cells responding to microbial antigens such as PC and LPS develop normally and are fully functional.

Elevation of immunoglobulin deposition in the synovial membrane of dogs with cranial cruciate ligament rupture.

Synovial membrane biopsies were collected from 32 dogs with spontaneous cranial cruciate ligament rupture and seven control dogs. The subclasses of immunoglobulins in the synovial membrane of both the diseased and control dogs were examined with immunohistochemistry in combination with image analysis. IgG deposition was four-fold higher and IgM was eight-fold higher in dogs with spontaneous cranial crucial ligament (CrCL) rupture. These findings support the theory that there is an immune component to the inflammation associated with canine CrCL rupture. Further investigation is required to determine if these immune or inflammatory responses in CrCL rupture joints are the primary factor in ligament rupture or if they develop secondary to ligament rupture and instability.