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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 19, 2004 as doi:10.1096/fj.03-1435fje. |
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Divisions of
* Immunotherapy,
Biochemical Immunology, and
Immunobiology, Research Center Borstel, Borstel, Germany; and
Helios Agnes Karll Hospital, Bad Schwartau, Germany
2Correspondence: Division of Immunotherapy, Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany. E-mail: sbrandau{at}fz-borstel.de
SPECIFIC AIMS
Recent evidence suggests that platelets are not only involved in haemostatic processes but also modulate immune responses. As antigen-presenting cells (APC) are of crucial importance for the regulation of immunity, in this study we wanted to define the role of platelet factor 4 (PF-4) as one of the major platelet-derived chemokines on the transition of monocytes into APCs.
PRINCIPAL FINDINGS
Human peripheral blood monocytes can be differentiated in the presence of GM-CSF or a combination of GM-CSF and IL-4 into macrophages and dendritic cells (DC), respectively. While the effects of GM-CSF on monocytes are well established, differentiation of monocytes under the influence of inflammatory stimuli has been rarely investigated and is not clear at present. We studied the effect of PF-4, a major inflammatory platelet-derived chemokine, and IL-4 on the differentiation, function, and immunostimulatory potential of human monocytes.
1. PF-4 in conjunction with IL-4 induces differentiation of monocytes into antigen-presenting cells clearly distinct from conventional macrophages and dendritic cells
In a first set of experiments we investigated the phenotype of PF-4/IL-4-differentiated monocytes. Our flow cytometric analysis revealed a phenotype of these PF-4/IL-4-differentiated monocytes that was clearly distinct from macrophages (GM-CSF) and DC (GM-CSF/IL-4) as well as macrophages differentiated in the presence of PF-4 alone (Fig. 1
). Stimulation with PF-4 and IL-4 for 7 days resulted in substantial expression of CD1b/c, CD86, CD209 (DC-SIGN), high amounts of MHC class II, and low but very reproducible expression of CD83 on the cell surface. Together with the absence of CD14 this phenotype strongly differs from the phenotype of macrophages generated in the presence of GM-CSF. PF-4/IL-4-monocytes also clearly differed from DC. While DC expressed high amounts of CD1a and BDCA-1 (CD1c), these antigens were absent after PF-4/IL-4 differentiation. At the same time PF-4/IL-4 monocytes expressed relatively high amounts of CD86 and BDCA-3, two antigens virtually absent on conventional DC. Expression of BDCA-3 is of particular interest, because this newly identified antigen has recently been described as a marker-antigen for a subset of rare human blood DC.
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Time-kinetic studies on PF-4/IL-4 stimulation of monocytes revealed additional differences between PF-4/IL-4 monocytes and DCs. PF-4/IL-4 stimulation led to a rapid down-regulation of CD14 together with an up-regulation of activation markers like CD83 and CD86, which were strongly expressed on day 4 of the differentiation process even in the absence of microbial stimulation. Between day 4 and day 7 of differentiation, PF-4/IL-4-stimulated monocytes, down-regulated CD83 and CD86 again, and underwent dramatic morphological changes resulting in strongly adherent cells with irregular shape and dendrite-like extrusions. Thus, our initial experiments identified a phenotype of these PF-4/IL-4-differentiated monocytes distinct from conventional macrophages and dendritic cells. In subsequent studies we referred to this cell type as PF-4/IL-4-differentiated antigen-presenting cells (PFAPC).
2. PFAPC are specialized APC that induce substantial lymphoproliferation and NK cytotoxicity but provoke only modest cytokine responses
In the second part of our study we assessed the function and immunostimulatory capacity of PFAPC. In particular, we investigated cytokine secretion by PFAPC, their phagocytic activity and interaction with NK cells as well as activation of lymphocytes in MLR reactions and antigen presentation assays. Because we observed phenotypic differences between day 3 PFAPC and day 7 PFAPC, we analyzed the function of PFAPC at these two time-points during the differentiation process.
Phagocytosis studies revealed that PFAPC, in contrast to macrophages, lost their ability to generate oxygen radicals. On the other hand, PFAPC retained their ability to take up particulate material and, in contrast to conventional DC, this ability was not reduced after LPS-induced maturation. We next analyzed the lymphostimulatory capacity of PFAPC and found that day 3 PFAPC were strong inducers of lymphoproliferation in MLR reactions (Fig. 2
A) and antigen presentation assays (Fig. 2B
) fully comparable to conventional DCs. In contrast, day 3 PFAPC induced only modest IFN-
production in lymphocyte cocultures (Fig. 2C
). On day 7 of differentiation PFAPC generally were less immunostimulatory compared with day 3 PFAPC (Fig. 2)
. Data obtained in PFAPC-lymphocyte cocultures (Fig. 2)
were substantiated by coculture experiments of PFAPC and NK cells. Day 3 PFAPC strongly enhanced cytotoxicity of NK cells against NK sensitive and largely NK resistant tumor cell lines. Lysis of target cells could be further augmented by microbial stimulation of cocultures with BCG mycobacteria. In sharp contrast, PFAPC mediated only minimal cytokine production in APC-NK cocultures even in the presence of stimulatory mycobacteria. Reduced induction of cytokine responses by PFAPC were further confirmed by direct comparison of the cytokine secretion by PFAPC and conventional DCs after stimulation with BCG, LPS, and CD154. These stimuli consistently induced lower amounts of TNF-
, IL-10, and IL12p70 in PFAPC compared with conventional DCs. These experiments suggest that PFAPC represent a specialized subset of antigen-presenting cells that induce substantial lymphoproliferation and NK cytotoxicity but provoke only modest cytokine responses.
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CONCLUSIONS AND SIGNIFICANCE
Professional antigen-presenting cells are of central importance for the initiation and outcome of immune responses under normal and pathological conditions. While the function of dendritic cells upon stimulation with inflammatory cytokines has been intensively investigated in the past, the effect of inflammatory mediators on the development and differentiation of APCs and dendritic cells is poorly understood. Platelet activation occurs under various pathological conditions and results in the release of large amounts of growth factors, chemokines, and cytokines. PF-4 is one of the most abundant chemokines released by activated platelets and to decipher the molecular and cellular interaction of this molecule with professional APCs therefore is of prime importance for our understanding of immune responses under pathological conditions and at sites of platelet activation.
By exposing monocytes to platelet-derived chemokine PF-4 in conjunction with IL-4 and employing morphological, immunophenotypical and functional parameters in this study we could demonstrate transition of monocytes into a unique specialized APC (Fig. 3
). Although these PFAPC shared certain features with mature dendritic cells, they were clearly distinct from both macrophages and conventional dendritic cells. We propose PFAPC as a subpopulation of specialized APCs generated under inflammatory conditions. According to our data these specialized cells would actively take up particulate material at sites of platelet activation and preferentially induce proliferation of lymphocytes while they only modestly alter the existing cytokine milieu. Thus, we have defined a new role for platelet-derived chemokine PF-4 as it can modulate immune responses by influencing the differentiation of human APCs from monocyte precursors. Future studies will more precisely define the influence of PF-4 on human APCs under pathophysiological conditions in vivo.
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FOOTNOTES
1 These authors contributed equally. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1435fje;
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