Substance P induces activation,adherence and migration of equine eosinophils

The tachykinin, substance P (SP), affects eosinophil function by direct and indirect mechanisms and has been shown to cause equine eosinophils to adhere to vascular endothelium and to release cytokines that increase cell adherence. The aim of this study was to determine whether SP could act directly on equine eosinophils in vitro. Eosinophil activation was also compared in cells from normal ponies and those with insect hypersensitivity as SP may be released in the skin of hypersensitive animals. SP caused equine eosinophils to adhere, migrate and produce superoxide, although high concentrations were required to produce these effects [10 +/- 2% adherence, 45 +/- 20 cells/0.3 mm2 and 48 +/- 7 nmol (of reduced cytochrome C)/106 cells, respectively, at 3 x 10-4 m]. That the 7-11, but not the 1-7, amino acid fragment of SP caused superoxide production, suggested the effects of SP were receptor mediated. Eosinophils from hypersensitive ponies produced more superoxide in response to SP, but not phorbol myristate acetate or histamine, over the concentration range tested when compared with cells from normal ponies. The data obtained in this study suggest that although SP can directly activate equine eosinophils, in view of the high concentrations required, such actions may be of less relevance physiologically than other SP-mediated effects.     

Cloning,expression and biological activity of equine interleukin (IL)-5

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.     

Uptake of 5-hydroxytryptamine by equine digital vein endothelial cells: inhibition by amines found in the equine caecum

REASONS FOR PERFORMING STUDY: 5-hydroxytryptamine (5-HT; serotonin) is a potent vasoconstrictor of equine digital blood vessels and has been implicated in the pathogenesis of acute laminitis. OBJECTIVES: The aims of this study were firstly to examine whether cells of the digital blood vessel wall exhibited an active uptake mechanism for 5-HT and to characterise its efficiency; and secondly, to study the potential inhibitory effect on this process of other amines, produced in the equine caecum. METHODS: Confluent monolayers of equine digital vein endothelial cells (EDVEC) and equine digital vein smooth muscle cells (EDVSMC) were incubated with [3H]5-HT (0.1-250 micromol/l) and the total and active uptake calculated. Equine pulmonary vein endothelial cells (EPVEC) were used as a positive control. RESULTS: Both EDVEC and EDVSMC showed uptake of [3H]5-HT by nonfaci litated diffusion; however, only EDVEC showed evidence of saturable facilitated uptake mechanism, with a Km of 41.6 +/- 9.3 micromol/l, which was significantly higher than that of EPVEC (9.9 +/- 2.1 micromol/l). All 6 caecally-derived amines examined (tyramine, spermine, isoamylamine, tryptamine, phenylethylamine and isobutylamine) inhibited the total uptake of [3H]5-HT in a concentration-dependent manner, tyramine having the lowest IC50 (3.7 x 10(-6) mol/l). CONCLUSIONS: These data suggest that facilitated uptake into the endothelium could play a role in moderating the vasoconstrictor response to 5-HT in the equine digital circulation. POTENTIAL CLINICAL RELEVANCE: The vasoconstrictor action of 5-HT could be potentiated by gut-derived amines, providing a feasible link between GI disturbances and the pathophysiology of laminits.     

The effect of leukotriene B4, leukotriene C4, zymosan activated serum, histamine, tabanid extract and N-formyl-methionyl-leucyl-phenylalanine on the in vitro migration of equine eosinophils.

The migration of equine eosinophils under agarose in response to inflammatory mediators, an arthropod extract and a synthetic peptide was examined. A chemotactic index (CI) was calculated by determining the ratio of the distance of eosinophil migration towards the chemoattractant to the distance migrated towards a buffer. Differences between the CI of those eosinophils exposed to chemoattractants and those exposed only to buffer were assessed by an analysis of variance. All agents except leukotriene C4 and the buffer induced statistically significant directional migration of eosinophils. Leukotriene B4 (LTB4) was the most effective chemotaxin for equine eosinophils. Migration of eosinophils stimulated by 10(-9) M LTB4 exceeded that induced by concentrations of histamine six orders of magnitude greater. The response of equine eosinophils to inflammatory mediators was similar to the reported behavior of human eosinophils. The ability of tabanid extract to attract equine eosinophils suggests that arthropod induced tissue eosinophilia many not depend entirely upon immunological mechanisms. The peptide, N-formyl-methionyl-leucyl-phenylalanine attracted equine eosinophils at 10(-4) M and 10(-3) M, concentrations that exceed those reported to be stimulatory for eosinophils of other species. The results of this study indicate that equine eosinophils are capable of migrating towards diverse stimuli, of which LTB4 was the most effective. It is plausible that LTB4 figures prominently in equine inflammation, particularly in lesions dominated by eosinophils.     

Role of the chemokine eotaxin in the pathogenesis of equine sweet itch

The chemokine eotaxin is involved in the recruitment of eosinophils and T helper 2 lymphocytes in human allergic diseases, and drugs that block its activity, including eotaxin receptor (CCR3) antagonists, are being developed. The authors have recently cloned the horse ortholog of eotaxin and shown that it can induce equine eosinophil migration and activation in vitro. Moreover, eotaxin mRNA expression was upregulated in cultured horse dermal fibroblasts exposed to equine interleukin-4, suggesting a possible source of this eosinophil chemoattractant in equine skin. The results of this study show that eotaxin and monocyte chemoattractant protein (MCP) 1, but not MCP-2 or MCP-4, mRNA expression is upregulated in skin biopsies of sweet itch lesions when eosinophils are present, when compared with clinically normal skin from the same ponies.     

Protein kinase C (PKC) isotype profile in eosinophils from ponies with sweet itch and role in histamine-induced eosinophil activation

Eosinophils have been implicated in the pathogenesis of the seasonal equine allergic skin disease, sweet itch. Protein kinase C (PKC) is involved in regulating eosinophil function and antigen challenge has been reported to alter PKC isotype expression in blood eosinophils from allergic human subjects. Here we have compared the pattern of PKC isotype expression in eosinophils from sweet itch ponies with that in cells from normal ponies both during the active and inactive phases of the disease. A role for PKC in histamine-induced eosinophil activation was also investigated. Conventional PKCs alpha and beta, novel PKCs delta and epsilon and atypical PKCs iota and zeta were identified in eosinophils pooled from four allergic ponies during the inactive phase, when no clinical signs were evident. The PKC isotypes, like those in eosinophils from normal ponies, were located primarily in the particulate fraction of the cell. Isotype expression in cells from normal and allergic animals did not appear to be different. In contrast, during the active phase of the disease, when the sweet itch ponies had clinical signs, the expression of PKCs beta, epsilon and iota in eosinophils from these animals appeared to be increased relative to that in cells from normal ponies. When PKC expression in eosinophils from five individual normal and sweet itch ponies was compared, small, but statistically significant, increases in PKC epsilon and PKCdelta expression were evident in eosinophils from the sweet itch ponies during the active and inactive phases, respectively. The non-selective PKC inhibitors, staurosporine and Ro31-8220, significantly reduced histamine-induced superoxide production. Use of G?6976, an inhibitor of conventional PKCs, suggested that PKCalpha and/or beta were involved and that there was significantly greater inhibition of the response in eosinophils obtained from sweet itch ponies during the active phase. There was no significant difference in histamine-induced superoxide production by eosinophils from allergic and normal ponies and the functional significance of the increased PKC isotype expression in eosinophils from sweet itch ponies relative to that in cells from healthy animals remains to be established.     

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity)

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.     

Inhibition of histamine release from dispersed canine skin mast cells by cyclosporin A, rolipram and salbutamol, but not by dexamethasone or sodium cromoglycate

Despite the important role that canine skin mast cells play in IgE-mediated allergic inflammation, clinically useful compounds for modulating mediator release from these cells or for suppressing cell response are lacking in the dog. The ability of five compounds to inhibit histamine release induced by non immunological (calcium ionophore A23187 and substance P) and IgE-dependent (concanavalin A) stimuli were compared. Sodium cromoglycate, a mast cell stabilizer, and dexamethasone, a glucocorticoid, failed to inhibit histamine release from isolated skin mast cells following any kind of stimulation. Salbutamol, a β-adrenergic agonist, exhibited inhibitory activity (46.0%) only after concanavalin A activation. In contrast, rolipram, a selective phosphodiesterase IV inhibitor and cyclosporin A, an immunosuppressor, showed potent anti allergic actions, inhibiting both IgE-dependent and -independent stimuli. Rolipram inhibited 42.8%, 44.7% and 19.2% of the mediator release induced by ionophore A23187, substance P and concanavalin A, respectively. Similarly cyclosporin A induced 85.9%, 14.9% and 67.3% inhibition after ionophore A23187, substance P and concanavalin A stimulation, respectively. These results suggest that rolipram and cyclosporin A merit to be clinically tested as agents for the treatment of chronic allergic diseases in the dog.     

Cloning of equine chemokines eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, mRNA expression in tissues and induction by IL-4 in dermal fibroblasts

We report the cloning of four equine CC chemokines, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, which show high levels of identity with their respective homologous sequences in other species. Using a multiplex RT-PCR, we have studied the constitutive mRNA expression of these four CC chemokines in skin, lung, liver, spleen, jejunum, colon and kidney of normal adult horses and compared this data with the eosinophil counts in the same samples. We demonstrate that eotaxin mRNA is only expressed in jejunum and colon, where there are large numbers of eosinophils suggesting that eotaxin might be recruiting eosinophils in the normal digestive tract of the horse. MCP-1 and MCP-4 are expressed in all tissues whereas MCP-2 is only found in some samples of lung, spleen, liver and kidney. We also report the early induction (2h) of equine eotaxin and MCP-4, and the up-regulation of MCP-1 by interleukin-4 in dermal fibroblasts, suggesting these chemokines might be involved in equine skin allergic diseases.     

Role of intracellular kinases in the regulation of equine eosinophil migration and actin polymerization

Inappropriately activated eosinophils can contribute to disease pathogenesis and intracellular signalling pathways that regulate functional responses may represent a therapeutic target. Little is known about intracellular signalling in equine eosinophils and this study examined the role of phospholipase C (PLC) and a range of protein kinases on responses to histamine and CCL11. Histamine (10(-4) M) or CCL11 (5.6 x 10(-9) M)-induced actin polymerization, migration and superoxide production by eosinophils from healthy horses were compared in the presence and absence of selective kinase inhibitors. Inhibition of phosphatidylinositol-3 kinase (PI3K) significantly reduced the response in each assay. In contrast, whilst inhibition of PLC decreased actin polymerization and superoxide production, an increase in migration was observed; the latter effect was also seen when protein kinase C (PKC) was inhibited. With the exception of histamine-induced migration, which was significantly reduced by blocking extracellular regulated kinase (ERK)1/2, activation of ERK1/2, p38 MAPK and tyrosine kinase did not appear to play an important role in the responses studied. These results suggest that equine eosinophil activation by histamine and CCL11 is mediated through PI3K. Whilst PLC activation is required for actin polymerization and superoxide production, migration may be negatively regulated by PLC and PKC. These kinases represent potential targets for modulating eosinophil activation by multiple stimuli.     

Eosinophils of the horse: Part I: Development,distribution, structure and biochemical mediators

Eosinophils are becoming the target of increasing research interest as recent studies suggest that their role in immune homoeostasis and the immune response to disease is far more complex than previously understood. Historically, the horse eosinophil has been used to study basic eosinophil biology because of the considerable volume of blood required to obtain enough viable cells for reliable, repeatable experiments. This resulted in a large but disseminated body of literature pertaining to the structure and function of the horse eosinophil. More recently, equine clinicians have produced case reports and clinical studies in an effort to define the role of the eosinophil in diseases of the horse. A thorough review of the equine eosinophil incorporating both bench research and clinical reports does not exist. The objective of this two-part review is to fill this need by integrating the basic science and clinical research into a comprehensive body of work on what is known specifically about the horse eosinophil, and its role in equine health and disease. Part I summarises the development and tissue distribution of eosinophils in the normal horse, and presents what is known about the cell structure, migration and biochemical mediators of the horse eosinophil. Part II reviews the role of the eosinophil in diseases of the horse, and concludes with a summary of knowledge gaps and open research questions to benefit both those who wish to use the equine eosinophil as a model for basic science research, and those whose primary interest lies with diseases of horse.     

Evaluation of an in vitro degranulation challenge procedure for equine pulmonary mast cells.

Pulmonary mast cells (PMC) are important components of the inflammatory process in equine allergic lung diseases such as heaves. Very little, however, is known of the degranulation kinetics of these cells and thus, their pathophysiologic role remains largely speculative. The purpose of this study was to develop a repeatable protocol for in vitro equine PMC degranulation. Five mature horses (sex: 2 M, 3 F; age: 8.8 +/- 6.5 y), historically free of pulmonary disease and normal on clinical respiratory examination, arterial blood gas analysis, pulmonary mechanics testing and histamine inhalation challenge, were studied. Bronchoalveolar lavage was performed on 4 separate occasions, at least 2 d apart, in a different lung lobe on each occasion. The lavage fluid was concentrated by centrifugation. Cells were resuspended in modified HEPES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC concentration determined. Cell inocula containing 30,000 PMC were incubated with 10(-8) to 6 x 10(-5) M A23187. Cells were then separated by centrifugation and histamine release (HR) was determined by fluorometric assay. The procedure was readily performed and yielded sufficient PMC for 30 to 60 inocula per lavage. Maximal HR (34.4 +/- 16.1%) was obtained with 10(-5) M A23187. The degranulation process was largely complete by 20 min but cell lysis was negligible. The challenge was repeatable within horse and produced a mean coefficient of variability of 23.0% following 20 min incubation with 10(-5) M A23187. We conclude that equine PMC degranulation can be repeatably performed in vitro and speculate that this protocol may be useful in further studies on the pathophysiology and treatment of equine allergic lung diseases.     

Effects of cyproheptadine and cetirizine on eosinophilic airway inflammation in cats with experimentally induced asthma

OBJECTIVE: To determine whether oral administration of cyproheptadine or cetirizine blocks the action of serotonin and histamine, respectively, and results in diminished eosinophilic airway inflammation in cats with experimentally induced asthma. ANIMALS: 9 cats in which asthma was experimentally induced through exposure to Bermuda grass allergen (BGA) during a 3-month period. PROCEDURES: Cats were randomized to receive monotherapy with each of 3 treatments for 1 week: placebo (flour in a gelatin capsule, PO, q 12 h), cyproheptadine (8 mg, PO, q 12 h), or cetirizine (5 mg, PO, q 12 h). A 1-week washout period was allowed to elapse between treatments. Prior to and following each 1-week treatment period, blood and bronchoalveolar lavage fluid (BALF) samples were collected. The percentage of eosinophils in BALF was evaluated to determine treatment efficacy. Serum and BALF BGA-specific immunoglobulin contents and plasma and BALF histamine concentrations were determined via ELISAs. Plasma and BALF serotonin concentrations were measured by use of a fluorometric method. RESULTS: The mean +/- SD percentage of eosinophils in BALF did not differ significantly among treatment groups (placebo, 40 +/- 22%; cyproheptadine, 27 +/- 16%; and cetirizine, 31 +/- 20%). Among the treatment groups, BGA-specific immunoglobulin content and histamine and serotonin concentrations were not significantly different. CONCLUSIONS AND CLINICAL RELEVANCE: In cats with experimentally induced asthma, cyproheptadine and cetirizine were not effective in decreasing airway eosinophilic inflammation or in altering several other measured immunologic variables. Neither cyproheptadine nor cetirizine can be advocated as monotherapy for cats with allergen-induced asthma.     

Plasma histamine concentration and histamine detection in peripheral blood eosinophils in cats

Plasma histamine levels were measured in 11 clinically healthy cats and 15 cats with allergic dermatitis. Histamine levels were markedly elevated in 5/15 allergic cats. A calcium ionophore, A23187, stimulates histamine release from feline peripheral blood cells. Immunostaining of blood smears from clinically healthy cats revealed that approximately 10% of eosinophils possessed histamine-containing granules. These results indicate that some peripheral eosinophils in cats contain histamine and can release histamine by appropriate stimulation.     

Distribution of CCR3 mRNA expression in horse tissues

CCL11 (also known as eotaxin) is a very potent and selective mediator of eosinophil migration which exerts its effects through its receptor, CCR3. In this study we report the cloning of an equine CCR3 cDNA sequence and investigation of the localization of CCR3 mRNA expression in horse tissues. Equine CCR3 displayed high levels of sequence identity with CCR3 sequences in other species. RT-PCR analysis revealed the expression of CCR3 in colon, lung and spleen of normal horses. In situ hybridisation experiments indicated that expression of CCR3 mRNA in colon was predominantly in eosinophils and to a lesser extent in mast cells, whereas CCR3 was seen mainly in lymphocytes of the lung and spleen. In view of the role of CCR3 in the recruitment of cells into sites of allergic inflammation, equine-specific CCR3 sequence data and information on tissue localization will be of potential benefit in the development of CCR3-targeted anti-inflammatory therapies in the horse.     

Atopic NC/Nga mice as a model for allergic asthma: cytokine profiles and eosinophil productivity of bone marrow

In previous study, NC/Nga mice with experimentally induced asthma showed severe eosinophilia. To explore the mechanism, profiles of representative cytokines interleukin (IL)-4, IL-5, and interferon (IFN)-gamma were examined in bronchoalveolar lavage fluid. The level of only IFN-gamma was lower in NC/Nga mice than control BALB/c mice. Furthermore, bone marrow cell culture system under the presence of eosinopoietic cytokines, which induce the differentiation of progenitor cells into mature eosinophils, showed that a larger number of eosinophils differentiated from NC/Nga mice derived bone marrow cells than from control BALB/c mice. These results may imply the possibility that severe eosinophilia in the NC/Nga mice are attributable to lower production of IFN-gamma and higher eosinophil productivity of bone marrow cells.     

Eosinophils of the horse: Part II: Eosinophils in clinical diseases

Interpretation of eosinophilia in body fluids or tissues is often not straightforward. Eosinophil counts vary among clinically healthy individuals, and considerable overlap can occur between normal and affected animals in conditions such as allergic airway disease. Parasite exposure is a confounding factor when counts are increased, and in cases where very high counts and dramatic clinical signs make another disease process obvious, the underlying pathology may be uncertain and treatment difficult. Eosinophils are a component of the immune response in many diseases of the horse, but their specific role is often unknown and likely multifactorial. In helminth infections, eosinophils are assumed to be part of the normal host response to a pathogen, whereas in multisystemic eosinophilic epitheliotropic disease (MEED), the predominance of eosinophils likely represents a wildly dysregulated response, or an abnormal response altogether. This distinction is still not clear for other diseases. Understanding the pathways involved in recruitment, activation or suppression of eosinophils is required for more accurate diagnostics, effective therapeutics, and successful strategies for prevention of eosinophil associated diseases. Eosinophils of the horse: Part II reviews published observations on the eosinophil in clinical diseases of the horse. The behaviour of eosinophils in three common and relatively well-studied conditions is presented first, including gastrointestinal helminth infections, non-infectious respiratory disease, and insect bite hypersensitivity. The less common eosinophil-associated diseases such as eosinophilic disease confined to the intestine (EDCI) and MEED are considered, followed by a brief summary of the eosinophil in phycomycosis and neoplasia. In conclusion, a panoramic view of the equine eosinophil as presented in Parts I and II is placed in the larger context of current eosinophil research, and areas of study are identified that may improve our understanding of eosinophil biology in equine health and clinical disease.     

Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 1. Evaluation of cytological stains and the percentage of mast cells and eosinophils

OBJECTIVE: To compare a fast Romanowsky cytological stain (Diff-Quik) and Leishman's stain for the detection of mast cells in samples from the lower airways of racehorses, and to compare the proportion of mast cells and eosinophils in the total inflammatory cells in tracheal aspirate (TA) with those in paired bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses. PROCEDURE: Fifty-one paired TA and BAL samples were collected after treadmill exercise from 48 horses with poor racing performance. Two slides were prepared from each sample; one was stained with Diff-Quik stain and the other with Leishman's stain. Differential cell counts of eosinophils and mast cells were recorded from each slide. Comparison of the suitability of the stains for the detection of mast cells, and comparisons of eosinophil and mast cell percentages in TA and BAL samples were analysed using the non-parametric Wilcoxon matched pairs test. RESULTS: Percentages of mast cells were significantly higher in Leishman than in Diff-Quik stained slides in both TA (P = 0.03) and BAL samples (P < 0.0001). Mast cell percentages were significantly higher in BAL than in TA samples using Leishman's stain (P < 0.0001). There was no significant difference in eosinophil percentages between TA and BAL samples (P = 0.07). CONCLUSIONS: Fast Romanowsky type stains (for example Diff-Quik) are not appropriate for the detection of mast cells in samples from the equine lower respiratory tract. Therefore, a metachromatic stain that reliably identifies mast cells (for example Leishman's) should be used if evaluation of mast cells in lower respiratory tract is undertaken. Mast cells are predominantly found in the distal small airways and alveoli sampled with a BAL. In contrast, eosinophils appear to be evenly distributed in the lower respiratory tract. However, high percentages of eosinophils are occasionally found only in TA samples. We recommend that both a TA and BAL be used for the evaluation of eosinophils and mast cells within the equine lower respiratory tract.     

The effects of Strongylus vulgaris parasitism on eosinophil distribution and accumulation in equine large intestinal mucosa

REASONS FOR PERFORMING STUDY: Eosinophilic granulocytes have been associated with parasite or immune-mediated diseases, but their functions in other disease processes remain unclear. Cause and timing of eosinophil migration into the equine gastrointestinal mucosa are also unknown. OBJECTIVE: To determine the effects of intestinal parasitism on eosinophils in equine large intestinal mucosa. METHODS: Large intestinal mucosal samples were collected from horses and ponies (n = 16) from the general veterinary hospital population, ponies (n = 3) raised in a parasite-free environment, ponies experimentally infected with 500 infective Strongylus vulgaris larvae and treated with a proprietary anthelmintic drug (n = 14), and a similar group of ponies (n = 7) that received no anthelmintic treatment. Total eosinophil counts and eosinophil distribution in the mucosa were determined by histological examination. A mixed model analysis was performed and appropriate Bonferroni adjusted P values used for each family of comparisons. P<0.05 was considered significant. RESULTS: There was no difference in large intestinal mucosal eosinophil counts and eosinophil distribution between ponies infected with S. vulgaris and those raised in a parasite-free environment. Experimental infection with S. vulgaris, with or without subsequent anthelmintic treatment, did not change eosinophil counts, and counts were similar to those for horses from the general population. CONCLUSIONS: Migration of eosinophils to the equine large intestinal mucosa appears to be independent of exposure to parasites. Large intestinal mucosal eosinophils may have more functions in addition to their role in defence against parasites.     

Adherence of Mycoplasma hyopneumoniae to cell monolayers

This work was an attempt to develop an in vitro adherence model for Mycoplasma hyopneumoniae, using monolayers of human and porcine lung fibroblasts and porcine kidney cells. Mycoplasma hyopneumoniae grown in Friis mycoplasma broth was radiolabeled with 35[S]-methionine, washed, concentrated, and inoculated on the monolayers. After 15 minutes of centrifugation to facilitate adherence, monolayers were washed 3 times, dissolved with 0.1N NaOH, and suspended in scintillation liquid, and the radioactivity was determined in a liquid scintillation counter. Adherence, measured as a percentage of counts added, varied according to the mycoplasma strain and the cell line used. Comparison of strains J, 144L, and 232 of M hyopneumoniae revealed 7.5 +/- 5.9, 31.9 +/- 13, and 9.6 +/- 5% adherence to porcine kidney cells, respectively. Slightly different, but proportionally the same relationships were obtained with swine or human fibroblasts. Adherence was decreased slightly by repeated washings of the mycoplasma-treated cell monolayers; however, a plateau was reached, indicating irreversibility of the adherence process. Pretreatment of cell monolayers with nonlabeled organisms substantially blocked adherence by labeled organisms. Dilution of labeled organisms resulted in an increased proportion adhering. Therefore, it appears that the adherence was a receptor-dependent event. Treatment of the mycoplasmas with trypsin prior to the inoculation of monolayers resulted in a marked reduction in adherence. Treatment of the mycoplasmas with hyperimmune swine serum against M hyopneumoniae or normal swine serum resulted in 80 to 90% reduction of adherence; however, no inhibition occurred when mycoplasmas were treated with purified IgG from the hyperimmune serum.