Conclusiones basadas en el objetivo de investigación 1

SUBSEQUENT DEACTIVATION

Adhesion of macrophages plays a cmcial role in the initiation of inflammatory processes by influencing their number and function at inflammatory sites. Although accumulation of macrophages is one of the most typical pathological features in various forms of glomerulonephritis [Main et al. 1992], little is understood about the

mechanisms involved in macrophage adhesiveness within the glomerular

microenvironment. We showed that mesangial cells, but not other cells tested, release factors which induce macrophage detachment from various substrata. We identified that TGF-61 is one of the factors involved in this activity.

The inhibitory effect of mesangial cell-derived medium on macrophage adhesiveness can be explained by several possible mechanisms. Certain factors secreted from mesangial cells could reduce adhesiveness via cytotoxicity. However, our data showed that the decreased adhesiveness was reversible and that cell viabihty and morphology was unaffected by mesangial cell-derived medium. Mesangial cell-derived factors might directly interfere with interactions between macrophages and substrata, but the fact that the initial adhesion of macrophages was not affected by the mesangial cell- derived medium, excluded this possibility. Mesangial cells may modulate macrophage adhesiveness by suppressing expression of certain adhesion receptors. Indeed, our data implied the requirement of new protein synthesis for the adhesive ability of macrophages since; i) macrophages pretreated with mesangial cell-derived medium showed reduced initial adhesion, ii) this impaired adhesiveness gradually recovered to the normal level following the withdrawal of mesangial cell-derived medium, and iii)

cycloheximide, a protein synthesis inhibitor, delayed recovery of impaired adhesiveness [T. Siito, preliminary results].

Macrophages use various receptors to adhere to the endothelium, ECM or other cells [Fraser et al. 1993; Hogasen et al. 1995; Hughes et al. 1995]. TGF-61 has been shown to down-regulate expression of certain adhesion receptors including scavenger receptors and CR3 in macrophages [Bottalico et al. 1991; Hogasen et al. 1995]. We also investigated the effect of mesangial cell-derived conditioned medium on the expression of the 132-integrin LFA-1 (GDI 1 a/CD 18), CR3 (GDI lb/CD 18) and p i 50/95 (GDI 1 c/CD 18) by macrophages. Although our results were inconclusive, together with our present finding that TGF-31 produced by mesangial cells is a negative regulator for macrophage adhesiveness, inhibited expression of these adhesion receptors by TGF-Bl might explain the suppressive effect of mesangial cell- derived medium on macrophage adhesiveness.

Using the anti-TGF-61 neutralising antibody, we found that the contribution of TGF- Bl was partial, since an excess of anti-TGF-Bl antibody inhibited only 35 % of the activity of mesangial cell-derived medium. Furthermore, total activity of mesangial cell- derived medium was decreased by heat treatment, suggesting the involvement of other thermolabile molecules. IL-10 or TN F-a could be the candidates [van Lenten and Fogelman, 1992; Moore et al. 1993], but expression of these cytokines is not normally detected in SM43 mesangial cells [Kitamura et al. 1996]. Further investigation will be required to elucidate the unidentified components in mesangial cell-derived medium.

In this study, we utilised plastic plates coated with different FGM components which are found in the normal glomerulus. Mesangial cell medium significantly reduced adhesiveness of macrophages seeded on laminin, collagen type IV or basement membrane Matrigel as well as on plain plastic. Interestingly, the effect of mesangial cell-derived medium was most pronounced on macrophages adhered to plastic, rather

than those on ECM-coated plates. This difference may be explained by the enhancement of macrophage adhesiveness by cell-ECM interaction since expression of integrins may be upregulated by TGF-Bl [Wahl et al. 1993].

Adherence is a priming trigger for a wide range of monocyte/macrophage functions. For example, adhesion induces differentiation of monocytes to tissue macrophages, facilitates migration, phagocytosis, respiratory oxidative burst, and expression of certain cytokines and proto-oncogenes [Springer and Anderson, 1986; Haskill et al. 1988; Newman and Tucci, 1990; Shaw et al. 1990; Azavedo and de Souza, 1992; Xing et al. 1992]. Based upon these findings, adhesiveness, possibly, controls function of macrophages within the glomerulus. We, therefore, examined the relationship between macrophage adhesiveness and activity following the exposure to mesangial cell-derived medium. Compared to adherent cells, non-adherent macrophages showed reduced mitogenic activity and blunted expression of cytokines in response to EPS. These findings indicate that mesangial cell-derived factors, including TGF-Bl, induce detachment of macrophages followed by their blunted responses to a specific stimulus.

In the acute, reversible model of anti-Thy 1 nephritis in the rat, a transient accumulation of monocytes/macrophages is observed within 24 hours following injection of the anti- Thy 1 antibody [Kawachi et al. 1992a]. The increased number of glomerular macrophages is sustained up to day 7 and declines thereafter. At day 14, the majority of the inflammatory macrophages disappear from the glomerulus [Kawachi et al. 1992a]. The mechanism involved in this process may include trafficking of inflammatory macrophages to draining lymph nodes [Lan et al. 1993b]. In the acute model of anti-Thy 1 glomerulonephritis, upregulation of glomerular TGF-Bl is detected from day 4 and plateaus until at least day 14 [Okuda et al. 1990]. Interestingly, the anti-Thy 1 antibody re-injected at day 14 does not induce macrophage accumulation [Kawachi et al. 1992a]. Upregulation of TGF-Bl is, thus, closely correlated not with accumulation but with reduced macrophage retention in the

glomerulus. The negative effect of mesangial cell-derived factors including TGF-61 on macrophage adhesiveness may play a role in the clearance of activated macrophages from the nephritic glomeruli and, in part, participate in the recovery of the glomerulus from acute inflammation.

4.2. MESANGIAL CELL-DERIVED TGF-Bl INHIBITS