Phenotypic characteristics of human monocytes undergoing transendothelial migration

Abstract

. We found that monocyte migration led to an enhanced expression of CD11a, CD33, CD45RO, CD54 [intercellular cell-adhesion molecule (ICAM)-1] and human leucocyte antigen-DR. The most striking increase was observed for ICAM-1 when ECs were activated with tumour necrosis factor-α and interleukin-1α. The results of our study indicate the following: (1) there is a characteristic immunophenotype on the surface of monocytes after transendothelial migration; (2) this phenotype seems to be induced by interactions between monocytes and ECs; and (3) this change is enhanced by the pretreatment of ECs with cytokines. Taken together, the results suggest that local cytokine production activating ECs is sufficient to enhance monocyte migration and that this, in turn, can induce changes consistent with an activated phenotype known to be interactive between antigen-presenting cells and T cells. These results have implications for our pathogenetic insights into rheumatoid arthritis.

Introduction:

].

].

Aims:

model. We found that monocyte migration led to an enhanced expression of CD11a, CD33, CD45RO, CD54 (ICAM-1) and human leucocyte antigen (HLA)-DR. The most striking increase was observed for CD54 when ECs were activated with TNF-α and IL-1α. Our findings of increased CD54 expression on monocytes that migrated through endothelium pretreated with TNF-α and IL-1α might be useful in elucidating the mode of action of therapy with antibodies against TNF-α and IL-1 in RA and thus might lead to new avenues of RA therapy in future.

Methods:

] and then cultured. ECs in the third to fifth passage were used. Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats of healthy blood donors by centrifugation over gradients of Ficoll-Hypaque. PBMCs were prepared immediately before starting the experiments.

ECs per well were incubated overnight.

) were resuspended in fresh culture medium, layered on top of collagen gels with and without ECs and incubated at 37°C. The range of the incubation period was 15 minutes to 24 hours. Nonadherent cells (NAD) were harvested by washing twice with culture medium. Cells bound to the surface (BND) were enriched by washing each well twice with warm (37°C) Puck's EDTA, twice with warm (37°C) EGTA [0.5 mM EGTA in phosphate-buffered saline (PBS)] and once with cold (4°C) Puck's EDTA. Finally, for the recovery of those cells that had migrated into the collagen gels (MIG), 0.7 ml of a solution containing 0.1% collagenase, 1% (v/v) fetal calf serum and 50 mM Hepes buffer was added per well. The collagen gels were then minced gently with a pipette and incubated for 60 minutes at 37°C, after which the migrated PBMCs were removed by washing the wells twice with PBS. Each population (NAD, BND and MIG) was washed, resuspended in culture medium and counted under a microscope. In some experiments we studied monocyte migration into plain collagen gels. In these experiments no ECs were layered on the collagen gels; in other respects the experiments were performed exactly as described above.

In some experiments the EC monolayer was preactivated by incubation with TNF-α, IL-1α, macrophage inflammatory protein (MIP)-1α or interferon-γ (IFN-γ). To this end, the medium in each well was removed and the ECs were incubated for 5 hours at 37°C with or without the respective cytokines or chemokines (100 IU/ml TNF-α, IL-1α or IFN-γ, or 50 ng/ml MIP-1α). After this 5-hour pretreatment, each well was washed thoroughly and the migration assay was performed as described above.

per sample) were incubated at 4°C for 30 minutes. Cells were then pelleted and resuspended in 250 μl of PBS before analysis was performed on a flow cytometer. All results are expressed as the respective mean fluorescence intensity among CD14-positive cells. Because not only monocytes but also ECs express CD54 (ICAM-1), in the analyses of the expression of CD54 on monocytes by fluorescence-activated cell sorting, monocytes were defined by both the scatter profile and the expression of CD14. In addition, ECs, which are considerably larger, were excluded by size.

-tests were used for comparisons.

Results:

, the presence of an endothelium clearly facilitated the migration of PBMCs: after 30 minutes the percentage of PBMCs that had migrated was twice as high as that in the absence of ECs. After 2 hours, about 40% of the PBMCs could be recovered from collagen gels coated with an EC layer, whereas only 24% of PBMCs had migrated into plain collagen gels. Prolonging the incubation time to 24 hours allowed further migration of PBMCs only in the absence of ECs, but did not significantly increase the extent of EC-mediated migration.

. The expression of CD11a, CD33, CD45RO, CD54 and HLA-DR was significantly higher in MIG than in NAD. When compared with BND, these markers, and also CD45RB and CD62L, were significantly elevated in MIG. NAD, BND and MIG were incubated with collagenase for the same durations to control for possible cell activation by the collagenase treatment; the expression of adhesion molecules was similar to that on untreated cells.

We also studied the capacity for monocyte migration into plain collagen gels, that is, in the absence of an endothelium. No significant difference in surface marker expression was observed when migrated monocytes were compared with any other fraction.

= 0.016).

).

).

Discussion:

). Taken together, our results suggest that transendothelial migration induces the activation or maturation of monocytes.

). In contrast, MIP-1α pretreatment did not change the monocyte phenotype investigated here. In the light of enhanced migration through MIP-1α-prestimulated endothelium, these results suggest a dichotomy of cytokine/chemokine effects on migration compared with surface marker expression: ECs activated with TNF-α and IL-1α seem to lead to an upregulation of both monocyte migration and surface marker expression, whereas MIP-1α only enhances migration, a finding that is compatible with the chemotactic chemokine nature of MIP-1α. Alternatively, MIP-1α could have been trapped in the collagen gel, acting as a chemotactic gradient directly on monocytes rather than via ECs. Thus TNF-α and IL-1α seem to mediate different proinflammatory events from those mediated by MIP-1α.

].

]. This is consistent with the increase in cell migration found in our experiments and the altered expression of ICAM-1 on monocytes. Because the classic ICAM-1 counter-receptors LFA-1 and Mac-1 have not been detected on ECs, the existence of another ICAM-1 ligand, one that facilitates the transendothelial migration of monocytes, remains possible.

].

In summary, our findings indicate that monocyte migration is accompanied by changes in function-associated surface antigens and that TNF-α and IL-1α in particular increase the number of migrating monocytes and lead to an enhanced expression of certain surface markers involved in cell-cell interactions. These events might not only be partly responsible for the high inflammatory activity in RA synovium; they also suggest that ECs have a pivotal role in these processes and thus might constitute an important therapeutic target.

Introduction

].

].

It was the aim of the present study to perform a detailed characterisation of the immunophenotype of transendothelially migrated monocytes.

Cell cultures

]. The culture medium consisted of MCDB-M 104 glutamine (Gibco, Paisley, UK) supplemented with 20% (v/v) fetal calf serum (FCS), 24 μg/ml EC growth supplement (TC Laevosan, Vienna, Austria), 50 IU/ml heparin, 2 mM L-glutamine (Gibco), penicillin (100 IU/ml; Gibco) and streptomycin (100 μg/ml; Gibco). ECs in the third to fifth passage were used.

Preparation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats of healthy blood donors by centrifugation over gradients of Ficoll-Hypaque (Histopaque R, Sigma, Vienna, Austria). PBMCs were prepared immediately before starting the experiments.

Monocyte-EC binding and transendothelial migration

ECs per well were incubated overnight.

) were resuspended in fresh culture medium, layered on top of collagen gels with and without ECs and incubated at 37°C. The range of the incubation period was 15 minutes to 24 hours. Nonadherent cells (NAD) were harvested by washing twice with culture medium. Cells bound to the surface (BND) were enriched by washing each well twice with warm (37°C) Puck's EDTA, twice with warm (37°C) EGTA (0.5 mM EGTA in PBS) and once with cold (4°C) Puck's EDTA. Finally, for the recovery of those cells that had migrated into the collagen gels (MIG), 0.7 ml of a solution containing 0.1% collagenase (Sigma), 1% (v/v) FCS and 50mM Hepes buffer (Gibco) was added per well. The collagen gels were then gently minced with a pipette and incubated for 60 minutes at 37°C, after which the migrated PBMCs were removed by washing the wells twice with PBS. Each population (NAD, BND and MIG) was washed, resuspended in culture medium and counted by microscope. In some experiments monocyte we studied migration into plain collagen gels. In these experiments no ECs were layered on the collagen gels; in other respects the experiments were performed exactly as described above.

Pretreatment of ECs

In some experiments the EC monolayer was preactivated by incubation with TNF-α (Pharma Biotechnology, Hannover, Germany), IL-1α, macrophage inflammatory protein-1α (MIP-1α) or interferon-γ (IFN-γ) (all purchased from Serotec, Oxford, UK). To this end, the medium in each well was removed and the ECs were incubated for 5 hours at 37°C with or without the respective cytokines or chemokines (100 IU/ml TNF-α, IL-1α or IFN-γ, or 50 ng/ml MIP-1α). After this 5-hour pretreatment, each well was washed thoroughly and the migration assay was performed as described above.

Analysis of monocyte surface markers by dual colour flow cytometry

per sample) were incubated at 4°C for 30 minutes. Cells were then pelleted and resus-pended in 250 μl of PBS before analysis was performed on a flow cytometer (FACScan; Becton-Dickinson). All results are expressed as the respective mean fluorescence intensity among CD14-positive cells. Because not only monocytes but also ECs express CD54 (ICAM-1), in the analyses of the expression of CD54 on monocytes by fluorescence-activated cell sorting, monocytes were defined by both the scatter profile and the expression of CD14. In addition, ECs, which are considerably larger, were excluded by size.

Statistics

-tests were used for comparisons.

Transendothelial migration of PBMCs

, the presence of an endothelium clearly facilitated the migration of PBMCs: after 30 minutes the percentage of PBMCs that had migrated was twice as high as that in the absence of ECs. After 2 hours, about 40% of the PBMCs could be recovered from collagen gels coated with an EC layer, whereas only 24% PBMCs had migrated into plain collagen gels. Prolonging the incubation time to 24 hours allowed further migration of PBMCs only in the absence of ECs, but did not significantly increase the extent of EC-mediated migration.

Phenotypic analysis of monocytes capable of transendothelial migration

. The expression of CD11a, CD33, CD45RO, CD54 and HLA-DR was significantly higher in MIG than in NAD. When compared with BND, these markers, and also CD45RB and CD62L, were significantly elevated in MIG. NAD, BND and MIG were incubated with collagenase for the same durations to control for possible cell activation by the collagenase treatment; the expression of adhesion molecules was similar to that on untreated cells.

Phenotypic analysis of monocytes migrated into plain collagen gels

We also studied the capacity for monocyte migration into plain collagen gels, that is, in the absence of an endothelium. No significant difference in surface marker expression was observed when migrated monocytes were compared with any other fraction.

Effect of pretreatment of ECs on the transendothelial migration of monocytes

= 0.016).

Effect of pretreatment of ECs on the immunophenotype of migrated monocytes

).

).

Discussion

). Taken together, our results suggest that transendothelial migration induces the activation or maturation of monocytes.

]. Here we found an upregulation of CD45RO in the whole population of migrated monocytes; the significance of this finding is not clear and also merits further elucidation.

]; our observation of upregulated CD11a on monocytes after transendothelial migration is in line with these data.

], had a high expression of ICAM-1. These findings and our results therefore suggest that the immunophenotype of macrophages or monocytes recovered from inflammatory lesions is determined, at least in part, by the process of transendothelial migration.

). In contrast, MIP-1α pretreatment did not change the monocyte phenotype investigated here. In the light of enhanced migration through MIP-1α-prestimulated endothelium, these results suggest a dichotomy of cytokine/chemokine effects on migration compared with surface marker expression: ECs activated with TNF-α and IL-1α seem to lead to an upregulation of both monocyte migration and surface marker expression, whereas MIP-1α only enhances migration, a finding that is compatible with the chemotactic chemokine nature of MIP-1α. Alternatively, MIP-1α could have been trapped in the collagen gel, acting as a chemotactic gradient directly on monocytes rather than via ECs. Thus TNF-α and IL-1α seem to mediate different proinflammatory events from those mediated by MIP-1α.

Generally, the influence of ECs on monocytes could be explained in two ways: either such signals are provided by cell-cell interaction via pairs of receptors and counter-receptors during the process of migration, or, alternatively, ECs could secrete chemoattractants. However, in the latter case we would instead expect similar effects on the bound subpopulation as well as a higher percentage of EC-bound monocytes.

].

]. This is consistent with the increase in cell migration found in our experiments and the altered expression of ICAM-1 on monocytes. Because the classic ICAM-1 counter-receptors LFA-1 and Mac-1 have not been detected on ECs, the existence of another ICAM-1 ligand, one that facilitates the transendothelial migration of monocytes, remains possible.

].

], it is tempting to speculate that some CD33 high monocytes might differentiate into dendritic cells in inflammatory sites.

] and led to a clinical benefit in patients with early or subacute RA.

In summary, our findings indicate that monocyte migration is accompanied by changes in function-associated surface antigens and that TNF-α and IL-1α in particular increase the number of migrating monocytes and lead to an enhanced expression of certain surface markers involved in cell-cell interactions. These events might not only be partly responsible for the high inflammatory activity in RA synovium; they also suggest that ECs have a pivotal role in these processes and thus might constitute an important therapeutic target.

Abbreviations

APC = antigen-presenting cell; BND = population of cells bound to the surface; EC = endothelial cell; ICAM = intercellular cell-adhesion molecule; IFN-γ = interferon-γ; IL = interleukin; MIG = population of cells migrated into collagen gel; MIP = macrophage inflammatory protein; NAD = non-adherent cell population; PBMC = peripheral blood mononuclear cells; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; TNF-α = tumour necrosis factor-α.

Acknowledgements

This work was supported in part by a research grant from Solvay Pharma, Klosterneuburg, Austria.

Figures and Tables

< 0.05) differences between the percentages of cells migrated in the absence of endothelium and in its presence.

= 0.043.

Migration through endothelium increases CD54 expression on monocytes. Histograms show the CD54 mean fluorescence intensity (mfi) of monocytes that migrated through untreated endothelium (grey line in each panel), or endothelium pretreated with tumour necrosis factor-α (black line in middle panel) or interleukin-1α (black line in bottom panel). CD54 mfi of the nonadherent monocyte fraction is shown by a dotted line (top panel), isotype controls are shown by a thin black line in each panel. The experiment shown is representative of three independent experiments.

= 0.019.

Description of the ligands and functions of the surface markers studied

Surface marker expression on different monocyte populations

< 0.05) NAD compared with BND.

Changes in surface phenotypes of migrated monocytes

< 0.05) compared with cells that migrated through untreated ECs.

Keywords

• endothelium
• inflammation
• migration
• monocyte
• transendothelial migration