Cytokine expression in naïve and previously infected lambs after challenge with Teladorsagia circumcincta.

Infection of sheep with Teladorsagia circumcincta triggers an immune response with predominantly type-2 (Th2) characteristics, including local eosinophila, mastocytosis and increased mucus production. In order to better understand the protective immune responses elicited, we used RT-PCR assays to define the changes in expression levels of a range of cytokine transcripts in lymph nodes draining the ovine abomasum following a challenge infection with T. circumcincta. This study compared the changes in cytokine expression in the abomasal lymph node following challenge with T. circumcincta in naïve sheep (Group 2) and sheep immunised by a previous trickle infection (Group 3), in comparison to unchallenged naive sheep (Group 1). There was a significant up-regulation of interleukin-4 (IL-4), IL-5 and IL-13 in both the challenged groups compared to naïve individuals. There was also an up-regulation of IL-1beta, IL-6, IL-10, IL-18, transforming growth factor-beta1 (TGFbeta1) and tumour necrosis factor-alpha (TNFalpha) by day 5 after infection. IL-12p40 was found to be increased in the previously infected Group 3 animals by day 5 following challenge. By contrast, transcription of this cytokine was found to be reduced by day 10 following infection of Group 2 animals. Expression of IL-2 and Interferon-gamma (IFNgamma) did not significantly differ between the three groups.

Characterisation of lesional infiltrates of dendritic cells and T cell subtypes during primary infestation of sheep with Psoroptes ovis, the sheep scab mite.

Earlier studies of cattle and sheep have demonstrated that Psoroptes ovis infestations provoke an intense immunoinflammatory response dominated by eosinophils accompanied by a substantial infiltrate of lymphocytes. However, the kinetics of the lymphocyte response and the subtypes involved have not been characterised. We employed two groups of sheep to investigate the early (1-21 days) and later (21-63 days) infiltration of lymphocyte subpopulations and dendritic cells in primary infestations of sheep with P. ovis. Immunohistochemistry indicated that by 4 days after infestation numbers of CD4+ and CD45RA+ cells in lesional skin had increased significantly (P<0.03 and P<0.005, respectively) and that a significant increase in gammadelta T cells and dendritic cells (CD1b+) had occurred by 8 days (P<0.02 and P<0.01, respectively). Numbers of lymphocyte and dendritic cells declined from 49 to 63 days after infestation. Our observations suggest that mite-derived products exert a profound influence on the early recruitment of lymphocytes that may significantly influence the genesis of the adaptive immune response.

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation.

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.

Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited.

Mucosal mast cells (MMC) play an important role in the immune response against selected species of intestinal nematode. The kinetics with which different strains of inbred mice resolve infection with Trichinella spiralis correlates with their ability to mount MMC responses in the intestinal mucosa. Homologues of MMC that express and constitutively secrete abundant amounts of the granule chymase, mouse mast cell protease-1 (mMCP-1), can be generated in vitro from bone marrow cultures supplemented with interleukins-3 and -9, stem cell factor and transforming growth factor-beta1. Using the enhanced growth characteristics of these MMC homologues, a novel limiting dilution assay for mast cell precursor (MCp) frequency has been developed. The assay is highly specific, in that cultures containing mast cells are identified with mMCP-1 specific antibody, and almost three-fold more sensitive than previously published systems. MCp frequencies were compared in BALB/c and C57/BL10 strains of mice that, respectively, respond rapidly and slowly to infection with T. spiralis. MCp frequency (1/378 bone marrow cells) was significantly greater in BALB/c than C57/BL10 mice (frequency: 1/751). Similarly the rate of growth of MMC homologues and the production of mMCP-1 was significantly greater in BALB/c than in C57/BL10 bone marrow cultures.

Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.

BACKGROUND: The mucosal mast cell (MMC) granule-specific beta-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-beta1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated. OBJECTIVE: To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2. METHODS: MMC homologues were generated by culturing bone marrow cells in the presence of TGF-beta1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA. RESULTS: mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited ( approximately 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2. CONCLUSION: The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators.

Cutaneous hypersensitivity reactions to Psoroptes ovis and Der p 1 in sheep previously infested with P. ovis--the sheep scab mite.

We have previously shown that infestation with Psoroptes ovis induces an IgE response and intense tissue eosinophilia, typical of a Type I hypersensitivity response [Parasite Immunol. 22 (2000) 407]. Intradermal tests (IDSTs) suggest that there are also delayed and Arthus-type responses to this parasite. In order to study the nature of ovine cutaneous reactions to P. ovis, naïve controls and experimentally infested sheep (n = 5) were challenged intradermally with mite antigen. Challenge elicited immediate (P < 0.001) and delayed (P < 0.005) wheal reactions in sensitised sheep. At 6 (P < 0.02) and 30 h (P < 0.001) the predominant infiltrating cells were eosinophils. To explore the role of circulating antibodies, naïve sheep (n = 5) were subjected to Prausnitz-Kustner (PK) tests. These elicited immediate (P < 0.02) but not delayed wheal reactions. At 6 h eosinophils (P < 0.001) dominated the infiltrate. These results suggest that P. ovis allergens provoke an IgE-dependent immediate and late phase response and a cell-mediated eosinophil-rich delayed-type hypersensitivity response (ER-DTH).

Identification of an antigen from the sheep scab mite, Psoroptes ovis, homologous with house dust mite group I allergens.

Infestation of sheep with the ectoparasitic mite Psoroptes ovis, results in a severe allergic dermatitis. Currently, little is known about the allergens/antigens that stimulate the allergic response. We have isolated an 836-bp cDNA from a P. ovis cDNA library which displays strong homology to cysteine proteases and, in particular, to the group I house dust mite allergens Der p 1, Der f 1 and Eur m 1. The cDNA was expressed in Escherchia coli, fused to a hexahistidine tag and the recombinant protein (Pso o 1) purified using a nickel-affinity column. The recombinant Pso o 1 was tested for recognition by immunoglobulin (Ig)G and IgE in serum from P. ovis naïve and P. ovis infested sheep. Using Western blots, both classes of antibody to Pso o 1 were detected in postinfestation serum. In enzyme-linked immunosorbent assays, a pronounced IgG-antibody response to Pso o 1 was detected in five of five sheep 3 weeks postinfestation. The IgE-antibody response to whole mite extract was poor in four of five animals. However, a marked IgE response occurred in the fifth animal, and IgE anti Pso o 1 was detected in the serum.

Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.

BACKGROUND: Mucosal mast cells (MMC) play a central role in gut hypersensitivities and inflammation. They are morphologically, biochemically and functionally distinct from their connective tissue counterparts. Massive hyperplasia of MMC occurs 7-10 days after intestinal infection with nematodes but it has never been possible to replicate this phenomenon in vitro. OBJECTIVE: (1) To determine whether mouse bone marrow-derived mast cells (mBMMC) grown in the presence of transforming growth factor (TGF)-beta1 could develop over the same time frame (7-10 days) as MMC in parasitized mice. (2) To compare the early expression of surface receptors (integrins alphaE and beta7, c-kit and FcepsilonR) with that of the MMC-specific granule chymase mouse mast cell protease-1 (mMCP-1). METHODS: Mouse bone marrow cells were cultured in the presence of IL-9, IL-3 and Stem Cell Factor (SCF) with or without TGF-beta1. mBMMC were quantified after toluidine blue or Leishmans' staining. Expression of MMC-specific mouse mast cell proteases was analysed by ELISA, immunohistochemistry and RT-PCR. Surface antigen expression was characterized by flow cytometry and confocal microscopy. RESULTS: TGF-beta1 promotes the development of abundant MMC-like mBMMC from bone marrow progenitor cells with kinetics, which closely parallel that seen in vivo. mRNA transcripts encoding mMCP-1 and -2 are readily detectable by day 4 ex vivo in cultures grown in the presence of TGF-beta1. Between 30 and 40% and 75-90% of the cells in these cultures on days 4 and 7, respectively, have typical mast cell morphology, are c-kit+, FcepsilonR+, integrin alphaEbeta7+, and express and secrete abundant mMCP-1. The integrin alphaE subunit is coexpressed with mMCP-1. CONCLUSION: The kinetics of mMCP-1+/alphaE+ mBMMC development, regulated by TGF-beta1, are consistent with that seen in vivo in the parasitized intestine. The normally down-regulatory functions of TGF-beta1 in haematopoiesis are superseded in this culture system by its ability to promote the early expression of alphaE and mMCP-1.