Adhesion proteins on human microglial cells and modulation of their expression by IL1α and TNFα

~) INSTITUTPASTEUR/ELSEVIER Paris 1993

Res. Virol. 1993, 144, 47-52

Adhesion proteins on human microglial cells and modulation of their expression by ILia and TNFct G. S6bire, C. H6ry, S. Peudenier and M. Tardieu Laboratoire de Neurovirologie et Neuroimmunologie, Universitd Paris XI, UFR Kremlin-Bic~tre, 94276 Le Kremlin-Bic~tre Cedex SUMMARY Expression of adhesion proteins on human microglial cells was studied by immunocytochemistry. Both microglial cells and peripheral blood monocytes expressed t32 integrins and molecules of the immunoglobulin superfamily at similar levels whereas the expression of the [31 integrins (¢2-VLA (very late antigen), ¢4-VLA, ¢5-VLA, a6-VLA} was higher on microglial cells than on monocytes. Stimulation of microglial cells with interleukin-1~ and tumour necrosis factor-a, the main cytokines detected in HIVl-infected central nervous system (CNS), increased the microglial expression of c~I-VLA, intercellular adhesion molecule-I, vascular cell adhesion molecule-1 and ~2-LFA-1 (leukocytefunction-associated molecule-I) but not of aL-LFA-1. Such an induction of adhesion molecules could facilitate penetration of HIVl-infected monocytes into brain parenchyma and their adhesion to CNS cells, and could maintain a chronic inflammation during human immunodeficiency virus-1 (HIV1) encephalopathy.

Key-words: AIDS, Encephalopathy, Macrophage, adhesion protein, HIV; integrins, Ig, ILl=, TNF~, Immunocytochemistry, Microglial cells, Inflammation.

INTRODUCTION Brain macrophages, including the resident microglial cells and the monocytes infiltrating the blood-brain barrier, are implicated in the generation of the intra-CNS (central nervous system) immune response. They also play a central role in the physiopathology of human immunodeficiency virus-1 (HIV1) encephalopathy, since they are the main or the only CNS cell type infected by HIV1 (Watkins et al., 1990; Vazeux et al., 1987; Brinkmann et al., 1992; Peudenier et al., 1991a). However, within the brain of patients affected by HIV1 encephalopathy, the low rate of HIV l-infected cells contrasts with the extensive CNS lesions observed and suggests an indirect effect of HIVl-infected macrophages on surrounding uninfected cells (Price

Received October 11, 1992.

et al., 1988). In vitro, HIVl-infected monocytic cells are able to induce a cytotoxic effect on astrocytes and neurons either by secretion of soluble factors or after cell-to-cell contact involving adhesion proteins (Giulian et al., 1990; Lipton, 1992; Tardieu et aL, I992).

Adhesion proteins belonging to the i31 integrins, [32 integrins and immunoglobulin superfamily participate in many immune and inflammatory functions of macrophages. Thus, the 0rE-leukocytefunction-associated molecule-1/intercellular adhesion molecule-1 (%-LFA-1/ICAM-1) interaction is involved in antigen presentation, whereas receptors for complements (CR3, CR4) are implicated in phagocytosis (Altmann et al., 1989; Hynes, 1992). They also have a major role in the control of adhesion of monocytes to either neurons and astrocytes or

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48

to endothelial cells and thus in the control of mononuclear cell migration through the blood-brain barrier and CNS parenchyma (Sloan et al., 1992; Birsdall et ai., 1992). The expression of these molecules on CNS cells is still poorly studied and only scan( data is available (Akihyama, and McGeer, 1990; Wilcox et al., 1990; Frohman et al., 1989; Birsdall et al., 1992).

normal serum diluted in phosphate-buffered saline (PBS) and then incubated with the first monoclonal antibody for 18 h at 4°C (table I). Optimal dilution of primary antibodies was determined by testing serial dilutions on freshly isolated monocytes as positive controls. The subsequent techniques used for immunocytochemistry have been described previously (Peudenier et aL, 1991b).

We analysed, in vitro, the ability of microglial cells to synthesize integrins and proteins of the immunoglobulin superfamily and compared it to that of monocytes. We also tested the effect of cytokines, interleukin-I (ILl) and tumour necrosis factor--, (TNF',) on the expression of adhesion proteins.

Cellular stimulation To stimulate microglial cells, the supernatant was removed and replaced by fresh MEM containing 50 U/ml of human rTNFct (activity > 2.0 x 107 U/mg; Boehringer Mannheim ; Meylan, France) or of human rILla (activity > 1.0 × l0 TU/mg; Boehringer Mannheim) for 24 h. Cultures were then washed with PBS and fixed in acetone before testing.

MATERIALS AND METHODS RESULTS

Cultures enriched in brain-derived macrophages Methods used to perform brain-derived macrophage cultures have been previously described (Peudenier et al., 1991a). Briefly, primary neural cell cultures were first obtained from spinal cord and cortex of 8- to 12-week-old human embryos and brain-derived macrophages isolated by selective adhesion of ceils released after shaking of primary cultures. Eighty to 90 °70 of the isolated cells were microglial ceils expressing several specific markers of macrophages (Peudenier et al., 1991a). Both primary cultures and microglial cultures were maintained in Eagle's minimal essential medium (MEM) containing 6 g/I glucose, completed with l0 070 foetal calf serum (FCS), l 070glutamine and 1 070 penicillin/streptomycin. Isolation and cultures of human monocytes Human mononuclear cells from healthy seronegative donors were isolated on Ficoll hypaque (Lymphoprep, Nycomed; Oslo, Norway) gradient followed by selective adhesion on plastic plates (two cycles of adhesion, one 20 min and the second 30 min at 37°C). Monocytes recovered by scraping were resuspended in RPM 1-1640 containing 10 070 FCS, 1 070 glutamine, 1 070 penicillin/streptomycin and plated on glass slides for less than 8 h before testing.

Expression of adhesion proteins from integrin and immunoglobulin superfamilies on microglial cells and monocytes Microglial cells and monocytes expressed all the tested adhesion proteins (table II). ",4-VLA, ",5-VLA and ",6-VLA (very late antigens of the 131 integrin family) expression was higher on microglial cells than on monocytes. Since expression of adhesion molecules might be modified by the culture conditions, we performed two controls. First, no TNFct or I L l ' , was detected in supernatants of microglial cell cultures (Sdbire et al., 1993). Secondly, to analyse the effect of in vitro adherence on the expression of i31 integrins, monocytes were seeded on glass slides and tested after an adhesion period of either less than 8 h or 10 days. The level of 131 integrin expression on adherent monocytes remained stable (a2-VLA, ct4-VLA and ¢t5-VLA) or increased slightly (from 12 o70 to 39 °70 for Qt6-VLA) after 10 days of adhesion.

Modulation of the expression of adhesion proteins on microglial cells by ILl', and TNFa

Microglial cells and freshly isolated monocytes were fixed in acetone, incubated for 60 min with 75 070 human

After stimulation by either I L i a or TNFct, microglial cells retained their round or elongated shapes and kept the same rate of growth as unstimulated cells. As shown in table II, both I L i a and TNFa indu-

CNS FCS HIV! ICAM riLl LFA

LPS MEM PBS rTNFa VCAM VLA

Detection of adhesion proteins by immunocytochemistry

= = = = = =

central nervous system. foetal calf serum. human immunodeficiencyvirus type 1. intercellularadhesion molecule. recombinantinterleukin-l. leukocyte-function-associatedmolecule.

= = = = = =

lipopolysaccharide. minimal essential medium. phosphate-buffered saline. recombinant tumour necrosis factor-~. vascular cell adhesion molecule. very late antigen.

ADHESION PROTEINS ON HUMAN MICROGLIAL CELLS

49

Table I. Primary monoclonal antibodies used for immunochemistry. Adhesion proteins tested Dilution

Manufacturer

~1 integrin family CD29 CDw49a CDw49b CDw49d CDw49e CDw49f

([31-VLA) (ctl-VLA) (a2-VLA) (a4-VLA) (at5-VLA) (a6-VLA)

1:75 1:75 1:75 1:75 1:75 1:75

Immunotech Immunotech T-cell sciences Immunotech Immunotech Immunotech

([32-LFA- 1) (aL-LFA- 1) (CR3) (CR4)

I: 100 1:100 1:100 1:50

Immunotech Immunotech (*) Becton-Dickinson

1:400 1:400 1:75 1:75 1:75 1:75 1:400

Immunotech Realef Immunotech Immunotech Medarex Immunotech Becton-Dickinson

~2 integrin family CD 18 CDI la C D I lb CD 11 c

Immunoglobufin superfamily CD54 VCAM- 1 CD16 CD32 CD64 CD58 CD4

(ICAM- 1) (Fc-rRIII) (Fc~'RII) (FcyRI) (LFA-3) (Leu-3a)

(*) Gift from Dr. K. Pulford (J. Radcliffe Hospital, Oxford).

Table II. Expression of adhesion proteins on monocytes and microglial cells either kept unstimulated or stimulated with I L l a (50 U / m l ) or T N F a (50 U / m l ) . Adhesion proteins tested

Monocytes FCS

Labelled cells (°7o) Microglial cells FCS FCS + T N F a

FCS + ILa

~1 integrin family CD29 ([31-VLA) CDw49a ( a l - V L A ) CDw49b (~2-VLA) CDw49d (a4-VLA) CDw49e (cx5-VLA) CDw49f (a6-VLA)

78 + 24 48 +- 3 34 + 21 3 4 _ 13 47 ___29 12 + 1

67 + 25 35 + 5 44 + 30 57 + 13 90 + 7 85 ___9

ND 56 ___6 ND ND ND ND

ND 49 __+16 ND ND ND ND

87+3 80 ___4 89 ___4 85 + 7

64+2 59 + 7 85 +__17 4 9 + 12

9 0 + 1.5 60 + 5 ND ND

7 2 + 1.5 59 + 4.5 ND ND

66+23 19+ 11 88 ± 5 84 ± 11 82 ± 3 66 +__5 44+- 5

66+3 37 + 6 68 + 16 68 + 41 95 + 8 73 + 1 < 1

88+4 54+_ 5 ND ND ND ND ND

81 + 10 71 + 0 . 2 ND ND ND ND ND

~2 integrin family CD18 ([32-LFA-1) C D I la (aL-LFA- 1) CD1 lb (CR3) C D I lc (CR4)

Immunoglobulin superfamily CD54 (ICAM-1) VCAM-1 C D I 6 (Fc~'RIII) CD32 (FcyRII) CD64 (FcyRI) CD58 (LFA-3) CD4 (Leu-3a)

The data presented are the mean + SD of 2 to 3 experiments. Peripheral blood monocytes were kept for less than 8 h in culture on glass slides. Microglial ceils were kept for 7 days in culture on glass slides,

50

G. St~BIRE E T AL.

Fig. 1. Microglial cells either unstimulated (a) or stimulated Co) with ILl,, (50 U/ml) and labelled with an ICAM-I monoclonal antibody by immunocytochemistry. After stimulation with ILl% nearly all microglial cells were stained and the staining increased on each cell. Arrows indicate areas of staining.

ced increased expression of all adhesion proteins tested except 0tL-LFA-1, which remained at the same level of expression. This was especially intense for ICAM-I : nearly all microgliai cells became strongly stained by anti ICAM-1 antibody (fig. 1).

DISCUSSION

Comparative study of the pattern of expression of adhesion proteins on microglial cells and on peripheral blood monocytes demonstrated that both

ADHESION

PROTEINS ON HUMAN

MICROGLIAL

CELLS

51

cells expressed adhesion molecules from [31 integrins and the immunoglobulin superfamily, and confirmed the expression of [32 integrins on microglial cells, as already shown on brain tissue from Alzheimer patients (Akihyama and McGeer, 1990). These results also t~einforced the hypothesis of a monocytic origin of microglial cells.

Acknowledgements

131 integrins appear to be more strongly expressed on microglial cells than on monocytes, which was unexpected, since the previously studied expressions either of antigenic determinants (CD4, CD14, Ki-M6, Ki-M8) or of cytokines (ILl[3 and TNFct) were lower on microglial cells than on monocytes, which suggests a restriction of competencies during maturation from monocytes to microglia (Peudenier et al., 1991a; Peudenier et al., 1991b; S6bire et al., 1993). [31 integrin overexpression does not seem to be simply induced by glass-adherence of microglial cells during the culture, since freshly isolated monocytes kept adherent for l0 days expressed similar (0t4-VLA, ~,5-VLA) or only slightly increased (0t6-VLA) levels of [31 integrins. Upregulation of [31 integrin expression might be a characteristic of microglial phenotype and depend either on a genetically determined program of differentiation or on environmental factors produced by adjacent neuro-ectodermal cells.

Expression des molecules d'adh6sion/t la surface des cellules microgliales: modulation de leur expression par I'ILla et le TNFu

To evaluate the effect of cytokines on the microglial expression of adhesion proteins, we chose to test IL10t and TNFQt, which are involved in the regulation of many steps of the interactions between HIV1 and target cells (Matsuyama et al., 1991), and are detected in HIV 1 encephalopathy on CNS tissue sections (Tyor et al., 1992). Moreover, in vitro, human embryonic microglial cells stimulated with LPS were the main or the only CNS cells able to produce ILl (S6bire et al., 1993). Stimulation by ILI~ or TNF~ increased the expression of all of the adhesion molecules tested (ICAM-1, VCAM-1, [32-LFA-1, ctlVLA) except 0tL-LFA-1. The discrepancy between the increased expression of the [32 chain shared by [32 integrins and the stability of the 0tL chain of LFA-1 suggests upregulation of another member of the [32 family, such as CR3 and/or CR4. Upregulation of complement receptors could correspond to an increase in microglial scavenging functions in HIV1 encephalopathy. A cascade reaction during HIV1 infection might start with the secretion of cytokines such as ILl by microglial cells acting in an autocrine manner to upregulate adhesion protein synthesis. Contact between newly synthesized [31 integrins and proteins of the extracellular matrix could retain brain macrophages at the inflammatory sites, triggering proliferation and inflammatory mediator secretions, as demonstrated for blood monocytes and lymphocytes fflynes, 1992).

We thank A. Fischer for helpful discussion and L. Outin for photographic assistance.

L'expression des prot6ines d'adh6sion/~ la surface des cellules microgliales est 6tudi6e par immunocytochimie. Les cellules microgliales comme les monocytes fra2chement isol6s du sang p6riph6rique expriment toutes les prot6ines d'adh6sion test6es, ce qui constitue un nouvel argument en faveur de l'origine monocytaire de la cellule microgliale. Le niveau d'expression des [32-int6grines et des molecules appartenant/l la superfamille des immunoglobulines est identique/l la surface de la microglie et des monocytes. Par contre, les [31-int6grines (~2-VLA, 0t4-VLA, 0t5-VLA et a6-VLA) pr6sentent un niveau d'expression plus 61ev6/L la surface des cellules microgliales que des monocytes. La stimulation des ceilules microgliales par l'ILla (interleukine la) ou par le TNFcz (tumour necrosis factor a) - principales cytokines d6tect6es au sein du syst~me nerveux central infect6 par le VIH1 - - augmente l'expression de al-VLA, ICAM-1, VCAM-1 et de [32-LFA-1. Une telle induction d'expression des mol6cules d'adh6sion pourrait participer au maintien d'un &at d'inflammation chronique d616t6re pour le tissu nerveux infect6 par le VIHI. Mots-clds: SIDA, Enc6phalopathie, Macrophage, VIH, Prot6ine d'adh6sion; ILia, TNF0t, Int6grines, Ig, Immunocytochimie, Inflammation, Cellules microgliales.

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