ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.
ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o. ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.
P91. PHENOTYPE AND ACTIVATION STATE OF T AND NK CELLS IN PORCINE COLOSTRUM Karolina Hlavova, Hana Stepanova, Martin Faldyna Department of Immunology, Veterinary Research Institute, Brno, Czech Republic; hlavova@vri.cz The distribution and activation markers of T cell subpopulations and NK cells in porcine colostrum were studied. The results indicate that the absolute number of lymphocytes in colostrum is the highest in the first day of lactation. The most predominant subpopulation were cytotoxic T cells followed by CD4+CD8+ double positive T cells, CD2+CD8+ γδ T cells and NK cells. Helper T cells and other γδ T cell subpopulations were present in colostrum in very low percentages. The study of the distribution of T cell subpopulations during the first 3 days of lactation revealed that the proportion remains constant, but the absolute numbers of T- and NK cells decrease significantly in the first hours of lactation. The expression of CCR7, CD11b, CD25, CD45RA and MHCII-DR was used to detect the activation state of T- and NK cells in colostrum. T-cell subpopulations showed central/effector memory phenotype suggesting that these are antigen experienced cells. NK cells showed high frequency of MHCII-DR+ cells. Acknowledgements The study was supported by the Ministry of Agriculture of the Czech Republic (MZE0002716202) and project AdmireVet (CZ 1.05/2.1.00/01.0006; ED0006/01/01). P92. DISTRIBUTION AND FUNCTION OF PORCINE MONOCYTES IN DIFFERENT LYMPHOID TISSUE AND THE LUNG DURING EXPERIMENTAL ACTINOBACILLUS PLEUROPNEUMONIAE INFECTION Petra Ondrackova, Lenka Leva, Zdenka Kucerova, Monika Vicenova, Marketa Mensikova, Martin Faldyna Department of Immunology, Veterinary Research Institute, Brno, Czech Republic; ondrackovap@vri.cz Monocytes play an essential role in the defense against bacterial pathogens. Bone marrow (BM) and peripheral blood (PB) monocytes in pigs consist of two main „steadystate“ subpopulations: CD14 hi /CD163 - /SLA-DR - and CD14 low /CD163 + /SLA-DR + . During inflammation, the subpopulation of “inflammatory” monocytes expressing very high levels of CD163, but lacking SLADR molecule (being CD14 low /CD163 + /SLADR - ) appears in the BM and PB and replaces the CD14 low /CD163 + /SLADR + subpopulation. However, the current knowledge of monocyte migration into inflamed tissues in pigs is limited. The aim of the present study was to evaluate distribution of “inflammatory” CD14 low / CD163 + /SLADR - monocytes during experimental inflammation induced by Actinobacillus pleuropneumoniae (APP), ability of these cells to produce cytokines and a possible role Analytical Cytometry VII 197
for chemokines in attracting “inflammatory” CD14 low /CD163 + /SLADR - monocytes into the tissues. Monocyte subpopulations were detected by flow cytometry. Chemokines and chemokine receptors were detected by RT-qPCR. Cytokines were detected by flow cytometry, imunohistochemistry, immunofluorescence and RT-qPCR. The “steady-state” CD14 hi /CD163 - /SLA-DR - and CD14 low /CD163 + /SLA - monocytes were found in the BM, PB, spleen and lungs of control pigs. After APP-infection, “inflammatory” CD14 low /CD163 + /SLADR - monocytes replaced the “steady-state” subpopulation in BM, PB, spleen and they, moreover, appeared in an unaffected area, demarcation zone and necrotic area of the lungs and in tracheobronchial lymph nodes. They did not appear in mesenteric lymph nodes. Despite the systemic inflammatory reaction after APP infection, BM and PB “inflammatory” CD163 + monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. The further analysis, however, showed that after in vitro stimulation with LPS from APP either BM or PB “inflammatory” CD163 + monocytes were able to produce significant amounts of pro-inflammatory cytokine TNF-α. This ability was, however, altered in APP-infected pigs. Finally, it was shown in APP-infected pigs that, contrary to BM and PB CD163 + monocytes, CD163 + monocytes infiltrating the necrotic area of inflamed lungs and tracheobronchial lymph nodes produced significant amounts of TNF-α, IL-1β, IL-6 and IL-8. Levels of mRNA for various chemokines with their appropriate receptors were found to be elevated in BM (CCL3-CCR1/CCR5, CCL8-CCR2/CCR5, CCL19-CCR7), necrotic area of lungs (CCL3-CCR1, CCL5-CCR1/CCR3, CCL11-CCR3, CCL22/CCR4) and tracheobronchial lymph nodes (CCL3-CCR1) and therefore they could play a role in attracting monocytes into inflamed tissues. In conclusion, “inflammatory” monocytes migrate from BM into PB and spleen, and further into the inflamed tissue and draining lymph nodes. Various chemokines could drive this migration. Moreover, PB and BM CD163 + monocytes do not contribute to the elevated cytokine levels in plasma during APP infection. On the other hand, CD163 + monocytes are source of pro-inflammatory cytokines in the lung lesions and draining lymph nodes during APP infection. Acknowledgements This study was supported by grants of Czech Science Foundation (P502/10/P362), MZe 0002716202 and Ministry of Education, Youth and Sports of the Czech Republic (CZ.1.05/2.1.00/01.0006, AdmireVet). 198 Analytical Cytometry VII
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- Page 145 and 146: given cell type are missing and the
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for chemokines in attracting “inflammatory” CD14 low /CD163 + /SLADR - monocytes into the<br />
tissues.<br />
Monocyte subpopulations were detected by flow cytometry. Chemokines and chemokine<br />
receptors were detected by RT-qPCR. Cytokines were detected by flow cytometry,<br />
imunohistochemistry, immunofluorescence and RT-qPCR.<br />
The “steady-state” CD14 hi /CD163 - /SLA-DR - and CD14 low /CD163 + /SLA - monocytes were<br />
found in the BM, PB, spleen and lungs of control pigs. After APP-infection, “inflammatory”<br />
CD14 low /CD163 + /SLADR - monocytes replaced the “steady-state” subpopulation in BM,<br />
PB, spleen and they, moreover, appeared in an unaffected area, demarcation zone and<br />
necrotic area of the lungs and in tracheobronchial lymph nodes. They did not appear in<br />
mesenteric lymph nodes.<br />
Despite the systemic inflammatory reaction after APP infection, BM and PB<br />
“inflammatory” CD163 + monocytes did not express elevated levels of a wide range of<br />
cytokines compared to control pigs. The further analysis, however, showed that after in<br />
vitro stimulation with LPS from APP either BM or PB “inflammatory” CD163 + monocytes<br />
were able to produce significant amounts of pro-inflammatory cytokine TNF-α. This<br />
ability was, however, altered in APP-infected pigs. Finally, it was shown in APP-infected<br />
pigs that, contrary to BM and PB CD163 + monocytes, CD163 + monocytes infiltrating the<br />
necrotic area of inflamed lungs and tracheobronchial lymph nodes produced significant<br />
amounts of TNF-α, IL-1β, IL-6 and IL-8.<br />
Levels of mRNA for various chemokines with their appropriate receptors were found<br />
to be elevated in BM (CCL3-CCR1/CCR5, CCL8-CCR2/CCR5, CCL19-CCR7), necrotic area<br />
of lungs (CCL3-CCR1, CCL5-CCR1/CCR3, CCL11-CCR3, CCL22/CCR4) and tracheobronchial<br />
lymph nodes (CCL3-CCR1) and therefore they could play a role in attracting monocytes<br />
into inflamed tissues.<br />
In conclusion, “inflammatory” monocytes migrate from BM into PB and spleen, and<br />
further into the inflamed tissue and draining lymph nodes. Various chemokines could<br />
drive this migration. Moreover, PB and BM CD163 + monocytes do not contribute to the<br />
elevated cytokine levels in plasma during APP infection. On the other hand, CD163 +<br />
monocytes are source of pro-inflammatory cytokines in the lung lesions and draining<br />
lymph nodes during APP infection.<br />
Acknowledgements<br />
This study was supported by grants of Czech Science Foundation (P502/10/P362),<br />
MZe 0002716202 and Ministry of Education, Youth and Sports of the Czech Republic<br />
(CZ.1.05/2.1.00/01.0006, AdmireVet).<br />
198 Analytical Cytometry VII
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