Garry Barcham

Isolation and characterization of a novel eosinophil-specific galectin released into the lungs in response to allergen challenge

A novel galectin cDNA (galectin-14) was cloned from ovine eosinophil-rich leukocytes by low stringency reverse transcriptase-PCR and cDNA library screening. Data base searches indicate that this gene encodes a novel prototype galectin that contains one putative carbohydrate recognition domain and exhibits most identity to galectin-9/ecalectin, a potent eosinophil chemoattractant. The sugar binding properties of the recombinant molecule were confirmed by a hemagglutination assay and lactose inhibition. The mRNA and protein of galectin-14 are expressed at high levels in eosinophil-rich cell populations. Flow cytometry and cytospot staining demonstrate that the protein localizes to the cytoplasmic, but not the granular, compartment of eosinophils. In contrast, galectin-14 mRNA and protein were not detected in neutrophils, macrophages, or lymphocytes. Western blot analysis of bronchoalveolar lavage fluid indicates that galectin-14 is released from eosinophils into the lumen of the lungs after challenge with house dust mite allergen. The restricted expression of this novel galectin to eosinophils and its release into the lumen of the lung in a sheep asthma model indicates that it may play an important role in eosinophil function and allergic inflammation.

Isolation and characterization of a novel inducible mammalian galectin.

A novel mammalian galectin cDNA (ovgal11) was isolated by representational difference analysis from sheep stomach (abomasal) tissue infected with the nematode parasite, Haemonchus contortus. The mRNA is greatly up-regulated in helminth larval infected gastrointestinal tissue subject to inflammation and eosinophil infiltration. Immunohistological analysis indicates that the protein is localized in the cytoplasm and nucleus of upper epithelial cells of the gastrointestinal tract. The protein is also detected in mucus samples collected from infected abomasum but not from uninfected tissue. The restricted and inducible expression of ovgal11 mRNA and limited secretion of the protein support the hypothesis that OVGAL11 may be involved in gastrointestinal immune/inflammatory responses and possibly protection against infection.

Molecular cloning, expression and characterization of ovine TNFα

Tumour necrosis factor α (TNFα) is a cytokine with a wide range of effects on both lymphoid and non‐lymphoid cell types. By hybridization with a human TNFα cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNFα encodes a polypeptide of 234 amino acids that, based on analysis of human TNFα is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to the equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin‐D treated WEHI‐164 cells and induce COS cells to produce and secrete interleukin 6 (IL‐6). Further experiments demonstrated the importance of sequences within the 3’untranslated region of the cDNA in determining the level of expression of ovine TNFα Northern blot analysis was used to analyse the kinetics of induction of ovine TNFα mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNFα at 1 hand 5 h but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNFα mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNFα appears to exist as a single copy.

Recombinant cytokines as immunological adjuvants

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin‐2 (rovIL‐2), interleukin‐lα (rovlL‐lα) and tumour necrosis factor α. These purified proteins had specific activities in appropriate bioassays of 1 × 107, 1 times; 107 and 1 × 105 U/mg, respectively. Recombinant ovIL‐lα was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL‐1α significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 μg per sheep enhanced the humoral response to a similar extent. Recombinant ovIL‐1β had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL‐1 P into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Functional characterization of an eosinophil-specific galectin, ovine galectin-14

Across mammalian species, human galectin-10 and ovine galectin-14 are unique in their expression in eosinophils and their release into lung and gastrointestinal tissues following allergen or parasite challenge. Recombinant galectin-14 is active in carbohydrate binding assays and has been used in this study to unravel the function of this major eosinophil constituent. In vitro cultures revealed that galectin-14 is spontaneously released by eosinophils isolated from allergen-stimulated mammary gland lavage, but not by resting peripheral blood eosinophils. Galectin-14 secretion from peripheral blood eosinophils can be induced by the same stimuli that induce eosinophil degranulation. Flow cytometric analysis showed that recombinant galectin-14 can bind in vitro to eosinophils, neutrophils and activated lymphocytes. Glycan array screening indicated that galectin-14 recognizes terminal N-acetyllactosamine residues which can be modified with α1-2-fucosylation and, uniquely for a galectin, prefers α2- over α2-sialylation. Galectin-14 showed the greatest affinity for lacto-N-neotetraose, an immunomodulatory oligosaccharide expressed by helminths. Galectin-14 binds specifically to laminin in vitro, and to mucus and mucus producing cells on lung and intestinal tissue sections. In vivo, galectin-14 is abundantly present in mucus scrapings collected from either lungs or gastrointestinal tract following allergen or parasite challenge, respectively. These results suggest that in vivo secretion of eosinophil galectins may be specifically induced at epithelial surfaces after recruitment of eosinophils by allergic stimuli, and that eosinophil galectins may be involved in promoting adhesion and changing mucus properties during parasite infection and allergies.

Cysticercosis: cellular immune responses during primary and secondary infection.

Summary Immune reactions to cysticercosis have been extensively studied in mice. The lack of significant lymphocyte infiltration into the livers of infected mice and the obvious role of antibodies in rejection has led to the general conclusion that cellular reactions do not play a role in protection against this disease. In contrast, the present study examining the immune response to cestode infections in a large animal model (sheep) revealed the presence of a massive and highly organized cellular infiltration in the livers after a secondary Taenia hydatigena infection. The majority of the infiltrating lymphocytes were of the CD4+ phenotype with much fewer CD8+ cells present. While most γδ‐TCR+ cells in peripheral blood are SBU‐T19+, the majority of γδ ‐TCR+ lymphocytes in the liver lesions are SBU‐T19‐ suggesting selective migration of these cells into the lesions. In contrast to the diffuse distribution of T cells in the lesions, B cells were present as distinct aggregates.

Isolation, characterisation and expression of mRNAs encoding the ovine CC chemokines, monocyte chemoattractant protein (MCP)-1α and -2

CC chemokines are important mediators of immune responses, orchestrating the differential recruitment of various leukocyte populations. Despite the large number of known CC chemokines in other species, no cDNA encoding ovine CC chemokines have been isolated. A homology cloning strategy was utilised to isolate the cDNA of ovine CC chemokines. Full-length monocyte chemoattractant protein (MCP)-1alpha and -2 cDNA have been isolated. The predicted ovine MCP-1alpha amino acid sequence shares 87 and 75% identity with bovine MCP-1alpha and porcine MCP-1, respectively. The predicted ovine MCP-2 amino acid sequence shares 92 and 85% identity with bovine and porcine MCP-2, respectively. Northern blot analysis of MCP-1alpha revealed that it is strongly expressed in cells isolated from mammary lavage fluid (MAL) of ewes given intramammary infusions of Haemonchus contortus. Weak signals were detected in mammary and abomasal tissue. Southern blot analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products indicates that MCP-1alpha mRNA levels increase in abomasum after challenge with H. contortus. MCP-1alpha mRNA levels were also increased in bronchoalveolar lavage (BAL) cells and lung tissue after house dust mite extract (HDME) challenge. Similarly, MCP-2 mRNA was detected by Northern blot analysis at high levels in MAL cells after H. contortus intramammary infusion, and increased in BAL cells and lung tissue in HDME-challenged sheep. MCP-2 mRNA was not detected by Northern blots in whole mammary or abomasal tissue, but Southern blot analysis of RT-PCR products also indicates that MCP-2 mRNA increases in abomasal tissue after challenge with H. contortus. Hence, two ovine CC chemokine mRNA have been isolated that are up-regulated in response to parasite infection and allergen challenge. Ultimately the isolation of these and other ovine CC chemokines will help elucidate a wide variety of immune responses in sheep.

Parameters related to the application of recombinant ovine interleukin-1β as an adjuvant

This paper describes aspects of the safety and efficacy of recombinant ovine interleukin-1 beta (rovIL-1 beta) as an immunological adjuvant. A dose-response relationship was established using the intramuscular route, and significant adjuvant activity was observed following delivery of 10 or 100 micrograms of the cytokine delivered either in PBS or in combination with alum. Similar doses of rovIL-1 beta also showed adjuvant activity when delivered via the subcutaneous route. In experiments in both mice and sheep, rovIL-1 beta-mediated adjuvant activity was neutralised by a monoclonal antibody (mAb), 3.41, confirming that the adjuvant effect was due to the biological activity of the cytokine. Serum clearance rates and physiological responses to intravenous, intramuscular or subcutaneous administration of rovIL-1 beta in sheep were also determined. RovIL-1 beta was shown to have a serum half-life of 2 min. Transient body temperature increases of 2 degrees C following intravenous or subcutaneous delivery, or 1 degrees C following intramuscular delivery, were observed. White blood cell counts also fluctuated post-injection, which was shown to be due to changes in the number of circulating neutrophils. The action of the neutralising mAb on serum clearance, body temperatures and white cell counts was also determined. Co-injection of rovIL-1 beta with the mAb 3.41 prevented rapid clearance of the cytokine from the serum, and was associated with an extension in elevated body temperature. The mAb appeared to have no significant influence on the white blood cell profile induced following injection with rovIL-1 beta.

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

Background The Ca2+-activated K+ channel KCa3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of KCa3.1 modifies experimental asthma in sheep. Methodology and Principal Findings Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the KCa3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling. Conclusions These findings suggest that KCa3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of KCa3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production

Regulation of ovine alveolar macrophage function by recombinant interferon gamma (rIFN gamma) and lipopolysaccharide (LPS) was investigated. Ten units per millilitre of rIFN gamma increased surface expression of MHC class I and class II (DR alpha, DP alpha, and DQ alpha) molecules but not other surface antigens examined. The upregulation of MHC class II expression was specifically blocked by rIFN gamma specific monoclonal antibodies and determination of a dose/response curve established that the minimum concentration of rIFN gamma required for increased class II expression was 0.1 U ml-1 and for increased class I expression, 1 U ml-1. Northern blot analysis indicated that rIFN gamma mediated increases in surface MHC class I and class II expression were due to increased levels of specific mRNA. Using Northern blot analysis and homologous human cDNA probes we failed to detect mRNA encoding the cytokines IL-1 alpha, IL-1 beta, and TNF alpha in RNA extracted from freshly isolated macrophages or macrophages cultured in medium alone. Exposure of macrophages to LPS increased production of all three cytokines although kinetics of upregulation varied. TNF alpha mRNA was induced to maximal levels within 1 h, declining thereafter. IL-1 alpha mRNA was detected at 1 h post stimulation with a maximal level at 5 h, but none at 24 h. In contrast, IL-1 beta mRNA was not detected until 5 h after stimulation with a low level remaining at 24 h. Dose response analysis indicated that LPS concentrations of 100 pg ml-1 induced detectable levels of TNF alpha mRNA while levels as low as 10 pg ml-1 induced secretion of bioactive IL-1. Analysis of the kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages indicated that levels peaked at 24 h post stimulation.