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Comparative Study
. 2000 Jul;100(3):364-9.
doi: 10.1046/j.1365-2567.2000.00056.x.

Comparative analysis of integrin expression on monocyte-derived macrophages and monocyte-derived dendritic cells

C Ammon  1 , S P Meyer, L Schwarzfischer, S W Krause, R Andreesen, M Kreutz
Affiliations
Comparative Study

Comparative analysis of integrin expression on monocyte-derived macrophages and monocyte-derived dendritic cells

C Ammon et al. Immunology. 2000 Jul.

Abstract

Both macrophages (MAC) and dendritic cells (DC) are members of the mononuclear phagocyte system (MPS) with monocytes (MO) as common precursor cells. Cells of the MPS are able to take up, process and present antigens to T lymphocytes, thereby inducing a primary or secondary immune response. Adhesion molecules are of crucial importance for the interaction of antigen-presenting cells with immune cells, especially T lymphocytes. By representational difference analysis, we identified CD49c (VLA-3), a member of the beta1-integrin family of adhesion receptors, as differentiation-associated antigen in MO-derived MAC. In contrast, MO-derived DC did not express CD49c mRNA. These data prompted us to compare the integrin expression pattern of MAC and DC. Both cell types showed a low expression of the alpha-chains of the beta1-integrins CD49a, CD49b, CD49d and CD49e, whereas a marked difference was observed for CD49c and CD49f. Expression of both integrins increased during MO to MAC differentiation, but was not detectable on DC. In parallel the beta1-chain (CD29) was clearly up-regulated during MO to MAC differentiation but was only weakly expressed on DC. On the other hand, the beta2-integrins CD11a, CD11b, CD11c and CD18 were all expressed on MAC and DC. Beside their role in cell-cell interaction and adhesion, beta2-integrins are also known as possible binding molecules for bacteria and lipopolysaccharide (LPS), especially for high LPS concentrations. Therefore we investigated the LPS response of MAC versus DC in terms of tumour necrosis factor-alpha (TNF-alpha) release. DC were less responsive to low doses of LPS, which can easily be explained by the very low CD14 expression on DC compared for MAC. In contrast, the TNF-alpha response was comparable to MAC when DC were stimulated with high LPS concentrations. Our results show a specific, differentiation-dependent pattern of beta1- and beta2-integrin expression on in vitro-generated MAC and DC. We suggest that the high expression of CD11/CD18 on DC could be involved in the LPS binding of DC. As LPS is not only an activation but also a differentiation stimulus for DC, the expression of CD11/CD18 on DC may be important for the successful maturation of DC and thereby the initiation of a primary immune response.

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Figures

Figure 1
Figure 1
Expression of CD49c (VLA-3) mRNA during MO to MAC differentiation and in DC. (a) MO/MAC were harvested at the indicated time-points (day 0 – day 14), DC at day 7, and total RNA was prepared; 10 µg/lane were loaded and analysed for CD49c mRNA expression by Northern blot analysis. As an internal control the membrane was reprobed with an 18S rRNA oligonucleotide. One representative experiment out of three is shown. (b) Cells (1 × 105) were subjected to FACS analysis at the indicated time-points as described in the Materials and Methods. The mean fluorescence intensity (MFI) ± SEM out of at least three experiments is shown. The mean fluorescence intensity of the isotype control antibody was subtracted. (c) One representative flow cytometry experiment shows the surface expression of CD49c on macrophages and dendritic cells versus an isotype control antibody (IgG).
Figure 2
Figure 2
Expression of CD11c mRNA during MO to MAC differentiation and in DC. (a) Northern blot analysis was carried out as described in Figure 1; (b) for the CD11c mRNA expression in DC a longer exposure time of the film was needed to see the specific band clearly. For MO/MAC and DC one representative experiment out of three is shown. (c) FACS analysis was performed as described in the Materials and Methods. The mean fluorescence intensity (MFI) ± SEM out of at least three experiments is shown. The mean fluorescence intensity of the isotype control antibody was subtracted.
Figure 3
Figure 3
TNF-α production by MAC and DC after LPS stimulation. (a) MAC and DC were obtained by the culture conditions described in the Materials and Methods and harvested on day 7. After two washing steps cells were stimulated with the indicated amounts of LPS for 24 hr under serum conditions. The supernatant was analysed for the presence of TNF-α by cytokine ELISA. Data shown are the mean ±SEM out of four independent experiments. (b) Cells were obtained as described in (a) but stimulated for 24 hr under serum-free conditions. The supernatant was analysed for the presence of TNF-α by cytokine ELISA. Values given are the mean ±SEM for the same four donor cell populations as in Fig. 3(a).
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