Migration of polymorphonuclear leucocytes is influenced by dendritic cells

Abstract

Dendritic cells (DCs) are the most potent antigen-presenting cells and populate many tissues where they may participate in inflammatory reactions. The infiltration of polymorphonuclear leucocytes (PMNLs) into tissues is a prominent feature of inflammation. The mechanisms of PMNL recruitment depend on chemotactic factors and adhesion molecules expressed on endothelial cells. The aim of the present study was to determine whether DCs participate in the early recruitment of PMNLs. Dendritic cells derived from peripheral blood monocytes were used for this study. PMNLs incubated with culture supernatant (CS) from untreated or from tumour necrosis factor-alpha (TNF-alpha)-treated (1 hr, 100 U/ml, 37 degrees ) monocyte-derived DCs (moDCs) had increased surface expression of both CD11b and CD18. Moreover, both untreated and TNF-alpha-treated moDCs induced PMNL chemotaxis. By blocking CXCL8, CXCL5, CXCL7 and Pan GRO (CXCL1, CXCL2, CXCL3), we observed that CXCL8/interleukin-8 might be the chemokine that induced the PMNL chemotactic activity in the CS of untreated and TNF-alpha-treated moDC. Furthermore, we investigated the regulation of CXCL8 production in moDCs by adhesion molecule engagement. Our data demonstrated that CD31, CD18, CD29 and CD49d participated in the adhesion of immature moDCs to endothelium. Moreover, engagement of domains 1-3 of CD31, but not of CD29 or CD18, decreased the production of CXCL8 by immature but not mature moDCs (which display lower CD31 levels than immature moDCs). Overall, these results suggest that DCs not only trigger a specific immune response, but also the innate immune response by recruiting PMNLs. Furthermore, our results also suggest that CXCL8 production by immature DCs might be regulated by signalling through CD31 during their migration through the vascular endothelium.

Figure 1

Polymorphonuclear leucocyte (PMNL) activation (a) and migration (b, c and d). (a) CD18 and CD11b expression on PMNLs. The culture supernatant (CS) from monocyte-derived dendritic cells (moDCs) and from tumour necrosis factor-α (TNF-α) (100 U/ml, 1 hr at 37°)-treated moDCs was harvested after 3 hr, as described in the Materials and methods. Then, the leucocytes were incubated with the CS from moDCs and moDC-TNF-α for 10 min. Finally, PMNLs were stained with monoclonal antibodies (mAbs) to CD18 or to CD11b and analysed by flow cytometry. (b) PMNL migration induced by CS from moDCs and TNF-α (100 U/ml)-treated moDC harvested after 3 hr. Different dilutions of CS were added below the polycarbonate filters and the PMNLs were added above. They were incubated for 60 min, as described in the Materials and methods. The basal (▪) and fMLP (□)-induced PMNL migration is marked on the y axis. (c) PMNL migration induced by CS derived from moDCs treated for 1 hr with different concentrations of TNF-α (10–1000 U/ml). (d) PMNL migration induced by CS (diluted 1: 6) from unstimulated or from TNF-α-treated (100 U/ml) moDCs harvested at different time-points after moDC stimulation. In (a), one representative experiment of three is shown. For (b), (c) and (d), the results are expressed as the percentage of added PMNLs that migrated through the filter, and values represent the mean ± standard error of the mean (SEM) of three or four separate experiments performed in triplicate. **P < 0·01; *P < 0·05 (post hoc Student Newman Kules test).

Figure 2

Reverse transcription–polymerase chain reaction (RT–PCR) analysis for CXCL7 (lane 1), CXCL6 (lane 2), CXCL5 (lane 3), α-actine (lane 4) and CXCL8 (lane 5) mRNA in monocyte-derived dendritic cells (moDCs) and tumour necrosis factor-α (TNF-α)-treated moDCs. RNA was extracted from moDCs that were either treated or untreated with TNF-α.

Figure 3

Effect of monoclonal antibodies (mAbs) against CXCL8, CXCL5, CXCL7 and pan GRO on culture supernatant (CS)-induced polymorphonuclear leucocyte (PMNL) migration. CS were treated or not treated with the indicated mAb for 30 min at room temperature and then tested for their ability to induce PMNL migration. Data are expressed as the percentage of added PMNLs that migrated and represent the mean ± standard error of the mean (SEM) of four experiments performed in triplicate. **P < 0·01; ***P < 0·001 (post hoc Student Newman Kules test).

Figure 4

CXCL8 concentrations in monocyte-derived dendritic cells (moDCs), tumour necrosis factor-α (TNF-α)-treated moDC and lipopolysaccharide (LPS)-treated moDC culture supernatants (CS). (a) After stimulation with 100 U/ml of TNF-α or 100 ng/ml of LPS, the cells were incubated for 3 hr in RPMI-1640 containing 5 mg/mol human serum albumin (HSA). Then, the supernatants were recovered and diluted, and CXCL8 was assayed by enzyme-linked immunosorbent assay (ELISA). (b) Different dilutions of CS from moDC and CS from TNF-α-treated moDC. Data represent the mean ± standard deviation (SD) of one representative experiment of three. *P < 0·05; **P < 0·01; ***P < 0·001 (post hoc Student Newman Kules test).

Figure 5

Monocyte-derived dendritic cell (moDC) adhesion to human umbilical vein endothelial cells (HUVEC). [⁵¹Cr]-Labelled moDCs were preincubated for 20 min at room temperature with the indicated monoclonal antibody (mAb). Thereafter, moDCs were layered over unstimulated HUVEC monolayers for 30 min at 37°. Results are expressed as the percentage of added moDCs that were adherent. Data represent the mean ± standard deviation (SD) of one representative experiment of three. *P < 0·05; **P < 0·01 (post hoc Student Newman Kules test).

Figure 6

Effect of CD31, CD29 and CD18 ligation on CXCL8 production (a) and polymorphonuclear leucocyte (PMNL) migration (b) by monocyte-derived dedritic cells (moDCs). Untreated or lipopolysaccharide (LPS)-treated moDCs were cultured on immobilized CD31 monoclonal antibody (mAb) (5H2; 2B3), CD29 mAb (PAG11) or CD18 mAb (TS1/18) for 24 hr. Afterwards, the level of CXCL8 (a) and the chemotaxis ability of PMNL (b) in culture supernatant (CS) was determined. Data represent the mean ± standard error of the mean (SEM) of four independent experiments for (a) and of two for (b). **P < 0·01, *P < 0·05 (Dunnett multiple comparisons test).

Figure 7

Fluorescence-activated cell sorter (FACS) analysis of the DNA content of monocyte-derived DCs (moDCs) or of TNFα-treated moDCs after 24 hr of engagement by CD31 monoclonal antibody (mAb) or isotype-control mAb. moDCs were harvested, permeabilized and stained with propidium iodide. Cells per sample were analysed by FACS and cell counts were plotted against propidium iodide fluorescence. The percentage of cells of the hypodiploid (apoptotic) population is indicated on each graph. CV, coefficient of variation; Mn, mean.

Figure 8

Flow cytometric analysis of CD31 expression on untreated or on tumour necrosis factor-α (TNF-α)-treated monocyte-derived dendritic cells (moDCs). Cells were either untreated (solid line) or treated (dotted line) for 24 hr with TNF-α (100 U/ml) before staining with anti-CD31 monoclonal antibody (mAb) (clone 5H2). Controls consisted of untreated (filled histogram) or of TNF-α-treated moDCs (not shown owing to overlap) incubated with an isotype-matched irrelevant mAb. The data presented are representative of three independent experiments.

Figure 9

In vivo polymorphonuclear leucocyte (PMNL) migration induced by bone marrow-derived dendritic cells (BM-DCs) or by culture supernatant (CS) derived from BM-DCs. Murine air pouches were raised, and RPMI (200 µl), tumour necrosis factor-α (TNF-α) (10 ng/100 µl), untreated BM-DCs (5 × 10^5/100 µl) or tumour necrosis factor (TNF-α) (10 ng for 1 or 24 hr)-treated BM-DC were injected intrapouch (a). In another set of experiments, CS from untreated or from TNF-α-treated BM-DC were harvested and injected intrapouch (b). Two hours later, the mice were killed, the exudates collected and PMNLs counted by using a Cytoron flow cytometer. Data represent the mean ± standard error of the mean (SEM) of eight independent experiments for the results shown in (a) and of six independent experiments for the results shown in (b). ***P < 0·001, **P < 0·01, *P < 0·05 (Tukey-Kramer Multiple Comparisons test).