Harwood Academic Publishers imprint, part of the Gordon and Breach Publishing Group. Printed in Malaysia Defective Chemokine Production in T-Leukemia Cell Lines and its Possible Functional Role

Peripheral blood lymphocytes and T-cell clones produced nanogram quantities of the chemokines RANTES, MIP-lα, MIP-lβ, MCP-l, IL-8 and GRO-α as well as the motogenic cytokine HGF. In contrast, various T-leukemia cell lines at different stages of differentiation did not produce the same chemokines/cytokines. In order to study the possible functional importance of the poor chemokine production different T-cell lines were compared with respect to development of motile forms and migration on extracellular matrix components in the absence and presence of various chemokines. RANTES, MIP-1α, MIP-1β, IL-8, GRO-α and lymphotactin did not augment the development of motile forms including the size and appearance of the pseudopodia activity of the T-leukemia cell lines. The T-cell lines migrated spontaneously on/to fibronectin in a Boyden chamber assay system. Chemokines augmented the migration of the T-leukemia cell lines on fibronectin in the Boyden system in a chemotactic fashion with peak responses at 10 to 50 ng/ml. Thus, the production of chemokines is defective, in neoplastic T-lymphocytes. The defective chemokine production does not seem to play any major role for the basic locomotor capacity of the cells but may modulate the responsiveness to exogenous chemokines.

INTRODUCTION T-lymphocytes infiltrate tissues during inflammatory conditions of autoimmune and allergic origin and after neoplastic transformation as in Sezary's syndrome and Mycosis Fungoides (1)(2)(3). Since the capacity of T-lymphocytes to infiltrate plays a fundamental role in a variety of disease processes it is important to elucidate the regulation of T-cell infiltration. T-leukemia cell lines representing different stages of differentiation provide useful tools for this purpose. Furthermore, it is also important to elucidate whether motility per se or migration on tissue components is normal or altered in neoplastic conditions affecting T-cells. The present work focus on basic locomotor capacity and migration of T-leukemia cell lines on extracellular matrix (ECM) components.
The capacity of T-lymphocytes to migrate and infiltrate depends on the coordination of locomotor properties per se and adhesive interactions with endothelia and components of the extracellular matrix (ECM). T-cell infiltration comprises the adhesion of circulating lymphocytes to vascular endothelium followed by transendothelial passage and migration through the ECM (4)(5)(6). To enter sites of inflammation circulating lymphocytes use 2(LFA-1) and [1 (4 and o5) integrins to bind and migrate through endothelium that has been activated to express ICAM-1 and VCAM-1 by the local release of cytokines such as IL-1, TNF-cz or IFN-y (6,7). Additional targeting of T-cells to specific tissue sites is provided by receptors such as the cutaneous lymphocyte-associated antigen CLA which recognizes E-selectin on endothelial cells and is thought to be responsible for skin homing of T-cells (8).
During extravasation T-cells probably interact with ECM-components such as collagen type IV within the endothelial basement membrane (9-11) and after passage of the endothelium inflammatory T-cells migrate and accumulate in, an environment containing fibronectin (12)(13)(14). The extravascular migration of T-cells thus occurs in a milieu of ECM-proteins and probably involves sequential adhesion and deadhesion of lymphocyte [31-integrins with the matrix components (10,15). There is experimental evidence from in vitro models of lymphocyte migration that T-cells attach to and display motile behavior on ECM-components (16). Lymphocytes can thus migrate on the ECM-components FN, C IV and LM in a haptoand chemotactic fashion. This migration is mediated by [31-integrins which also together with 2-integrins function as triggering receptors for T-cell migration (10,17,18). Furthermore, there seems to be a functional specialization for T-cell migration to FN in that different T-cell lines use either 41 or c5[3for migration although they use both integrins for adhesion (16). The ability of T-lymphocytes to migrate through extravascular tissues towards a site of antigenic/inflammatory challenge is probably dependent on the ability of the cells to respond to a chemotactic gradient. The chemokines, a superfamily of small proteins (8000-14000 Mw) secreted primarily by leukocytes, are likely regulators of T-cell recruitment and infiltration during inflammation (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The superfamily of chemokines is classified as the C-X-C (), C-C (), C (y)-and the membrane bound CX3C (6)-groups, as defined by the spacing of the first two cysteines in a conserved four-cystein motif (30)(31)(32)(33)(34)(35). The chemokines probably influence both extravascular T-cell migration and the extravasation step per se. Thus, MCP-1 has been demonstrated to induce transendothelial T-cell chemotaxis (24,25). RANTES, MIP-I and MIP-I have been shown to be T-cell attractants using the Boyden microchemotaxis chamber (19,20,26,27). One plausible mechanism of action Of the chemokines is that they regulate T-cell adhesion. Thus, MIP-Iz, MIP-I, RANTES and IFN-y-inducible protein have been reported to augment adhesion of peripheral blood T-lymphocytes to recombinant endothelial adhesion molecules and to purified ECM-proteins (26,27). However, the recently described chemokines SLC (secondary lymphoid tissue chemokine) and Fractalkine seem to have even more profound effect on T-cell adhesion (31,32). Chemokines also regulate cellular polarization and adhesion receptor redistribution during interaction of activated blood lymphocytes with ICAM-1, VCAM-1 and 38 kd and 80 kd FN fragments (36)(37)(38)(39). This was suggested to represent a mechanism that enhances the recruitment of lymphocytes to inflammatory foci. T leukemia lymphocytes from patients exhibit motile behavior which seems to be pathologically elevated or depressed compared with that of normal T-cells (40,41). This suggests that there may be disturbances of motility in T-leukemia cells. However, it is difficult to analyze possible regulatory mechanisms such as interactions with endothelial cells, ECM-components or the influence of chemokines using patient material. T-leukemia cell lines representing different stages of differentiation (42) are useful alternative tools for comparative analysis of T-lymphocyte migration. Normal T-cell clones may constitute adequate reference cells for such analysis. Investigations of migratory properties of neoplastic T cell lines may thus elucidate the factors responsible for the abnormal migration behavior of T-leukemias and lymphomas.
Here we show that T-leukemia cell lines do not produce endogenous chemokines, while normal T-cell clones produce high levels of several chemokines and the motogenic cytokine HGF.

Cells
All T-leukemia cell lines used in these studies were purchased from American Type Culture Collection (ATCC, Rockville, MD). The birch-specific T-cell clone AF 24 was obtained from J. van Neerven, ALK Research Laboratory, HCrsholm, Denmark (AF 24). PBL were isolated as previously described (16).
Modified Boyden microchemotaxis chamber assay Lymphocyte migration was studied using a modified Boyden chamber assay system (Neuro Probe, Cabin John, MA). Briefly, a two chamber system, consisting of 48 wells, is devided by a filter with pores with a diameter large enough for cells to be capable to migrate through. Polyvinylpyrrolidone-free polycarbonate filters with 8 tm pore size (Porotics Co., Livermore, CA) were coated on the lower surface overnight with fibronectin 20tg/ml and subsequently washed in distilled water and air dried. The lower wells were filled with RPMI containing 10% FCS.
The upper wells were filled with 50 tl cells (2106 cells/ml) in RPMI with 10% FCS.
The chambers were incubated for 5 hours in a humidified incubator at 37C. Following incubation the filters were removed, fixed in methanol and stained with Giemsa (Reidel-de Ha6n, Seeize, Germany).
The filters were subsequently placed onto glass slides and remaining cells on the upper side of the filter were wiped off. The numbers of migrated cells were counted by light microscopy using a magnification of x200.
In order to analyze chemotactic effects of chemokines, chemokines were added at different concentrations to the medium in the lower wells. Each experiment was done in triplicate (three wells/concentration). The results are presented as mean values.

Chemokine production by normal and neoplastic T lymphocytes
The production of various chemokines by peripheral blood lymphocytes, T-cell clones and various neoplastic T lymphocyte cell lines representing different stages of T-cell differentiation was compared. The production of chemokines was determined in conditioned media using specific ELISA assays. It is evident from the ELISA assays shown in table I that the blood T-cells and a representative T-cell clone produced and released chemokines including RANTES, MIP-1 and , MCP-1, IL-8 and GROinto the culture medium. In addition, the motogenic cytokine HGF was detected in conditioned medium from the T-cell clone AF 24. Table I further shows that none of the different neoplastic T-cell lines produced any of the chemokines tested. We have also made several attempts to detect chemokines in conditioned media of neoplastic T lymphocytes after stimulation with forbol ester and cytokines (IL-2 and IL-4). All these attempts yielded negative results (data not shown). In conclusion, therefore normal T lymphocytes had a substantial chemokine production while neoplastic T-cells did not seem to produce chemokines. The T-leukemia cell lines showed dose-response curves to chemokines which usually had peak responses at 1-50 ng/ml. One T lymphocyte clone (AF 24) was tested and showed weaker migratory responses than the leukemic cell lines. Noteworthy, this T-cell clone showed peak responses at chemokine concentrations above 100 ng/ml (not shown).   (10,(19)(20)(21)(23)(24)(25)(26)(27)29) (data not shown). However, it can be seen in fig 2 that the chemokines did not augment the number of motile forms. Thus, the chemokines did not exert any obvious potentiating effect on the motile shape of the cells or on the development of pseudopodia. Furthermore, in some cases chemokines seemed to decrease the number of motile cells.

DISCUSSION
The major conclusion from the present study is that T-leukemia cell lines seem to have defective chemokine production. The data presented in this report thus establish that antigen-specific T-cell clones (table I) produce the chemokines RANTES, MIP-, MIP-, IL-8 and MCP-1 as well as the motogenic cytokine HGE In contrast, a number of leukemic T-cell lines did not produce chemokines The absence of chemokine production in T-leukemia cell lines represents a loss of a class of molecules implicated in the control of cell motility in comparison with "normal" T-cells.
However, in spite of the fact that the T-leukemia cell lines did not produce chemokines this defective chemokine production did not seem to influence the basic locomotor capacity of the cells .defined as development of motile forms and pseudopodia. Noteworthy, exogenous chemokines presented in the lower well of Boyden chambers promoted migration of T-leukemia cell lines showing that they possess capacity to respond to chemokines.
The T-leukemia cell lines showed peak migratory responses at lower chemokine concentrations than the "normal" T-cell clone. The difference in optimal chemokine concentrations for a motile response between separate cell types including normal and neoplastic T lymphocytes is not understood. Normal blood T-cells show maximal cytokine responses at 1 ng/ml and RANTES attract monocytes optimally at a concentration of 100 ng/ml (49). One possible explanation for the different dose-response profile to chemokines between T-leukemia cells and T lymphocyte clones may be that endogenous chemokines downregulate the chemokine responses of the normal cells by binding to chemokine receptors and that this binding either blocks or causes modulation/disappearance of the receptors. The literature contains numerous examples of up-and downregulation of growth factor/cytokine receptors by their ligands (26).
The present data point to the possibility that normal and neoplastic T lymphocytes may differ in their responses to chemokines although this needs further investigation. Such investigations should focus on the influence of continuous chemokine exposure on the expression of chemokine receptors by T-leukemia cells lacking endogenous chemokines. Does such ligand exposure modulate chemokine receptor expression per se or the sensitivity of chemokine receptors to their ligands? Understanding of possible differences in the mechanisms of chemokine action between normal and neoplastic T lymphocytes probably requires more experimental information concerning chemokine receptors in the same cells.
The fact that T-leukemia cells do not produce chemokines and other motogenic cytokines implies that they lack a regulatory system of endogenous mediators of migration which is present in normal T-cells. Normal T-cell clones, as observed in this study, can migrate chemotactically to their own chemokines. In non-lymphoid cell systems several autocrine motility factors have been described and characterized (50,51). These have been proposed to induce cell motility in normal situations such as healing and embryogenesis and to confer metastatic capabilities on neoplastic cells. However, a possible consequence of the lack of endogenous chemokines may be that T-leukemia cells display hyperresponsiveness to environmental chemokines.