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of the second sequence revealed a putative extracellular domain with 96% sequence identity to CD32; however, the predicted transmembrane and intracellular regions displayed 79% sequence identity to the Fc[gamma]RIII (CD16) molecule. This is of particular note as CD16, through noncovalent association with either the Fc[epsilon]RI gamma chain or the TCR zeta chain, acts to cause cell activation by means of ITAM motifs present in their cytoplasmic tails. We have detected soluble CD32 transcript in B cells, CD4+ and CD8+ T cells, CD14+ monocytes and neutrophils isolated from bovine blood. Transcript for the CD32/CD16 chimeric sequence was found in B cells, CD4+ T cells and CD14+ monocytes. Furthermore, cultured bovine B cells (BL3) were found to secrete a protein reactive with an antiCD32 monoclonal antibody (CCG32, IAH Compton). This is the first time a soluble FcR has been reported in a Species other than murine or human. The existence of these novel receptors adds a new level of complexity to cell regulatory functions involving IgG immune complexes.
number of cells expressing CD32 was greater than those expressing CD21. Mean fluorescence intensity for CD32 was significantly higher for adults (P < 0.01), indicating a greater number of CD32 receptors per lymphocyte compared to neonates. RT-PCR using RNA extracted from PBMCs of neonatal calves suggested production of a soluble version of Fc gamma RIIB (sCD32), lacking transmembrane region, which might contribute to lower membrane CD32 expression in neonates. Lower expression of CD21 in neonates combined with reduced levels of C3d in serum may be a limiting factor for activation of neonatal B cells. An age related increase in the percentage and absolute number of CD21 and CD32 positive lymphocytes, along with reduction in the level of maternal antibodies might help explain the ability of older calves to produce effective antibody responses compared to neonates.
doi:10.1016/j.vetimm.2008.10.215
Eleven porcine C gamma (C␥) genes: Phylogeny, expression and allotypic variants
Variation in expression of Fc gamma receptor IIB and CD21 on bovine lymphocytes with age K.S. Chattha Pathobiology, OVC, University of Guelph, Canada Species: Ruminants It is difficult to induce active immune responses in neonates, partly due to the limited functional ability of neonate’s immune system and partly due to inhibition mediated by maternal antibodies. Fc gamma receptor II B (CD32) is a major receptor responsible for suppression of antibody responses, through interaction with immune complexes of maternal antibodies (primarily IgG) and antigens, by virtue of its intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). CD21 (CR2), a receptor for complement component C3d, is expressed by B lymphocytes and binding results in lowering of the threshold for B cell activation. Thus it plays an important role in enhancing antibody responses to complement-associated antigens. CD21 (activating component), B cell receptor (membrane IgM) and CD32 (inhibitory component) form an intricate triad for determining the threshold of activation for B cells. Because cellular distribution of CD21 and CD32 of cattle has not been well documented, this study aimed to determine the variation in expression of CD21 and CD32 on bovine lymphocytes with age, particularly emphasizing differences between neonates and adults. Blood was collected from 20 healthy Holstein calves, 1–90 days of age, and 8 healthy adults. The percentage, absolute number and mean fluorescence intensity of CD21 and CD32 positive cells was determined using fluorochrome labelled monoclonal antibodies and flow cytometry. The percentage and absolute number of CD21 and CD32 positive lymphocytes increased with age from birth until about 60 days of age, with CD21 showing a greater percentage increase compared to CD32. The proportion of CD32 positive lymphocytes expressing CD21 also increased with age. In both calves and adults, the
doi:10.1016/j.vetimm.2008.10.216
John E. Butler 1,∗ , Nancy Wertz 1 , Nick Deschacht 2,3 , Serge Muyldermans 2,3 , Joan K. Lunney 4 1
Department Microbiology, University of Iowa, USA Cellular and Molecular Immunology, Vrije Universiteit Brussel, Belgium 3 Department of Molecular and Cellular interaction, VIB, Vrije Universiteit Brussel, Belgium 4 ANRI, ARS, USDA, Beltsville, MD, USA Keywords: IgG subclasses; Phylogeny; Fetus; IPP 2
E-mail address:
[email protected] (J.E. Butler). Species: Swine Subclass heterogeneity of swine IgG was recognized >40 years ago (Metzger and Fougereau, 1967; Kaltreider and Johnson, 1972), allotypic variation was described in 1982 (Rapacz and Hasler-Rapacz, 1982), differential specificity of anti-swine IgG antibodies has been recognized (Bokhout et al., 1986; Van Zaane and Hulst, 1987) and five C␥ gene sequences have been reported (Kacskovics et al., 1994). The full complexity of the system remains unresolved but we present here incremental data on eleven expressed C␥ genes which appear to comprise six subclasses. The allelic variants of IgG1 (IgG1a and IgG1b ) account for a very large proportion of the IgG transcribed in swine. This includes most of the IgG transcribed during fetal life. IgG2, also with two alleles, is transcribed at much lower levels as are IgG4, IgG5 and IgG6. IgG3 is a unique long-hinged variant which accounts for >50% of IgG transcripts in the ileal Peyers patches (IPP) and MLN of newborns but relative transcription declines as the IPP involute (Butler and Wertz, 2006). Sequence analyses confirm that speciation preceded subclass evolution so that a subclass with a certain name, e.g. IgG1, does not imply homology with those of the same name in other species. Therefore establishing structure–function relationships must be done for each species individually. Porcine IgG subclass proteins can-
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not be biochemically purified so porcine-camelid chimeric heavy chain antibodies (HCAb) have been constructed and expressed. Specificity tests failed to identify any subclassspecific monoclonal or polyclonal antibodies and showed most to be biased to the IgG1b allelic variant.
References Bokhout, B.A., van Asten-Noordijk, J.J., Stock, W., 1986. Mol. Immunol. 23, 675. Butler, J.E., Wertz, N., 2006. J. Immunol. 177, 5480. Kacskovics, I., Sun, J., Butler, J.E., 1994. J. Immunol. 153, 3536. Kaltreider, H.B., Johnson, J.S., 1972. J. Immunol. 109, 992. Metzger, J.J., Fougereau, M., 1967. C.R. Hebd. Seances Acad. Sci. Ser. D: Sci. Nat. 265, 724. Rapacz, J., Hasler-Rapacz, J., 1982. Proceedings of the 2nd World Congress on Genetics Applied to Livestock Production, vol. VIII. Editorial Garsi, Madrid. Van Zaane, D., Hulst, M.M., 1987. Vet. Immunol. Immunopathol. 16, 23.
doi:10.1016/j.vetimm.2008.10.217 9. Innate immunity, inflammation and adjuvants; memory, acquired immunity and vaccines Natural killer cells in lymph nodes of healthy calves express CD16 and show both cytotoxic and cytokineproducing properties Preben Boysen 1,∗ , Gjermund Gunnes 2 , Daniela Pende 3 , Mette Valheim 4 , Anne K. Storset 1 1
Department of Food Safety and Infection Biology, Norwegian School of Veterinary Sciences, Oslo, Norway 2 Department of Basic Sciences and Aquatic, Medicine, Norwegian School of Veterinary Science, Oslo, Norway 3 Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 4 Department for Animal Health, Section for Pathology, National Veterinary Institute, Oslo, Norway Keywords: Natural killer cells; Lymphoid organs; CD16; Cytotoxicity; Interferon-gamma E-mail address:
[email protected] (P. Boysen). Species: Ruminants Natural killer (NK) cells were recently shown to play an important immunomodulatory role in lymph nodes. We here report the presence, phenotype and function of NK cells resident in lymph nodes of several anatomical sites of healthy calves. NK cells, defined by NKp46 expression, were present in the paracortex and the medulla of bovine lymph nodes. Most lymph node-derived NK cells expressed CD16 and perforin, and a lytic capacity was demonstrated, while a well-developed interferon-gamma response to interleukin-2 and interleukin-12 stimulation was also seen. Lymph node-derived NK cells differed from those in blood by a higher expression of the activation markers CD44 and CD25, as well as CD8. Lselectin (CD62L) was expressed by the majority of lymph node-derived NK cells, consistent with a dependency of this molecule for migration to lymph nodes. Unlike in blood, the majority of lymph node NK cells had little or no CD2 expression. Compared to available literature, calf lymph nodes contained
NK cells in numbers equal to or higher than reported in humans, and clearly higher than in mice. These findings suggest a cytotoxic role of lymph node residing NK cells, beyond the predominantly cytokine-producing role previously inferred from studies on human NK cells. doi:10.1016/j.vetimm.2008.10.218 CD14+ cells are required for TLR7/8 ligand-induced IL-12 and IFN-␥ responses in bovine blood mononuclear cells Joram Buza 1,∗ , Ponn Benjamin 1 , Jianzhung Zhu 1 , Arthur M. Krieg 2 , Lorne A. Babiuk 1 , George K. Mutwiri 1 1
Vaccine & Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 1L1 2 Coley Pharmaceutical Group, Wellesley, MA 02481, USA Keywords: Innate immunity; CD14+; TLR7; TLR8; ssRNA; CpG; IL-12; IFN-␣, IFN-␥, TNF-␣, ELISA; ELISPOT; Bafilomycin; Cattle E-mail address:
[email protected] (J. Buza).
Species: Ruminants Toll-like receptors (TLR) link innate and adaptive immune responses. Single stranded viral RNA (ssRNA) was recently identified as the natural ligand for TLR7 and TLR8. ssRNA sequences from viruses, as well as their synthetic homologues stimulate innate immune responses in immune cells from humans and mice. In the present investigations, we tested whether synthetic ss oligoribonucleotides (ORN) can activate immune cells from cattle. In vitro incubation of bovine peripheral blood mononuclear cells (PBMC) with ORN-induced production of IL-12, IFN-␥ and TNF-␣. No significant induction of IFN-␣ was observed. Furthermore, depletion of CD14+ cells from the PBMC resulted in the loss of IL-12 and IFN-␥ response, confirming that CD14+ cells are required for cytokine response following stimulation with ORN. Since TLR7/8 are located in the intracellular compartment, and require phagosomal maturation for effective signalling, we tested whether ORN-induced responses could be influenced by inhibitors of phagosomal maturation. Pre-treatment of PBMC with bafilomycin (an inhibitor of phagosomal acidification) prior to stimulation with ORN abolished the cytokine responses, confirming that the receptor(s), which mediate the ORN-induced responses are intracellular. These results demonstrate for the first time that ORN have strong immune stimulatory effects in cattle, and suggest that further investigation on the potential of TLR7/8 ligands to activate innate and adaptive immune responses in domestic animals are warranted. doi:10.1016/j.vetimm.2008.10.219
