Monocyte‐derived dendritic cells from horses differ from dendritic cells of humans and mice

Abstract

SummaryDendritic cells (DC) are the initiators of immune responses and are present in most tissues in vivo. To generate myeloid DC from monocytes (MoDC) in vitro the necessary cytokines are granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Using degenerated primers delineated from other species and rapid amplification of cDNA ends reverse transcription-polymerase chain reaction (RACE RT-PCR), the cDNA of equine (eq.) GM-CSF was cloned and found to have a point deletion at the 3 0 -end of eq.GM-CSF, resulting in a 24-nucleotide extended open reading frame not described in any species thus far. For differentiating eq.MoDC, monocytes were stimulated with eq.GM-CSF and eq.IL-4. The eq.MoDC was analysed by both light and electron microscopy and by flow cytometry and mixed lymphocyte reaction. The eq.MoDC obtained had the typical morphology and function of DC, including the ability to stimulate allogeneic T cells in a mixed lymphocyte reaction. In contrast to the human system, however, monocytes had to be differentiated for 6-7 days before immature DC were obtained. Our data also indicate that lipopolysaccharide or poly(I:C) alone are not sufficient to confer the full phenotypic transition into mature DC. Thus our study contributes to understanding the heterogeneity of immunity and adds important information on the equine immune system, which is clearly distinct from those of mice or man.

An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: Divergent maturation programs by IL-4 and LPS

“…The two existing protocols used 1000 U/ml recombinant huGM-CSF together with 1000 U/ml ( ¼ 10 ng/ml) eqIL-4, used as a IL-4-transfected Chinese hamster ovary (CHO) cell supernatant, and reached a maximum of 8 Â 10 6 DC per 100 ml blood at day 3-6 of culture (Hammond et al, 1999). When both cytokines were used from an eq source lower cytokine concentrations of 250 U/ml eqGM-CSF plus 100 U/ml eqIL-4 were sufficient for DC generation, but the culture time was extended to 6 to 7 days and a maximum yield of 4 Â 10 6 DC per 500 ml blood was reported (Mauel et al, 2006). In our hands, performing the adherence step in AIM V s medium improved the cell yield by a factor of about 7 and additional use of Primaria s dishes by another factor of about 3, which led to a total increase of the DC yield by about 20-fold as compared with the standard plastic and medium used by the protocols published previously.…”

“…Culture periods for eqDC of 6-7 days were suggested by existing protocols (Hammond et al, 1999;Mauel et al, 2006). Since we detected substantial numbers of DC already at earlier time points, we analysed the expression kinetics of several surface markers related to DC after 2, 3, 4 and 7 days.…”

“…The generation of eqDC from monocytes (eqMoDC) succeeded with human GM-CSF and eqIL-4 (Hammond et al, 1999). Finally, eqDC generation was performed exclusively with eq cytokines (Mauel et al, 2006).…”

Analysis of CD14 Expression Levels in Putative Mesenchymal Progenitor Cells Isolated from Equine Bone Marrow

“…To determine if the equine CD14 antibody bound a protein of the expected size (40 kDa), immunoprecipitation and Western blot analysis was performed. A portion of the white blood cell (WBC) lysate was also immunoprecipitated using an antibody known to recognize human CD14 (mouse anti-human CD14, clone biG 10, catalog # 021-1c.2; Biometec, Griefswald, Germany) and previously validated for crossreactivity and specificity with equine cells [17,18] …”

The role of dendritic cells in alphaherpesvirus infections: archetypes and paradigms

“…Later on, this was improved by using recombinant equine IL-4 and recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) [68] or recombinant equine GM-CSF [69]. For studying DCs of cattle, two models are being used.…”