Transforming growth factor β and interleukin-1: a paradigm for opposing regulation of haemopoiesis

10 Transforming growth factor Il and interleukin-1: a paradigm for opposing regulation of haemopoiesis FRANCIS W. RUSCETII CLAIRE M. DUBOIS STEN E. W. JACOBSEN JONATHAN R. KELLER

Numerous studies have shown that the bone marrow has an organized and structured architecture in which close relationships exist between haemopoietic cells and the regulatory microenvironment (Lord, 1990). For example, stromal cells producing a stimulator(s) of haemopoietic progenitor cell proliferation (Lord et al, 1977) are in the highest concentration near the bone surface, where spleen colony-forming units (CFU-S), the stem cells that produce macroscopic col~nies in the spleens of irradiated recipients, are proliferating (Lord and Wright, 1984). By contrast, stromal cells that produce an inhibitor(s) of proliferation (Lord et al, 1976) are concentrated near the central axis of the bone where the more primitive, slowly cycling CFU-S are found. Increasing evidence supports the hypothesis that local production of inhibitory molecules ensures that few stem cells are in cell cycle during normal steady-state conditions. However, during the course of a cytoreductive treatment. levels of stimulators would be expected to rise and of inhibitors to decline. Then. when haemopoiesis recovers, levels of inhibitors would again rise, while those of stimulators would decline. The question remains a~ to t~e i~e~tity, number .and mecha.nism(s) of actio~ of cytokines invol~ed I~ rnaintammg ~t~m cells.m non-cycling to slowly cyc~mg to rapidly proliferating states. It IS interesting that many of the cytokines implicated in increasing stem cell cycling (interleukin (IL)-1. IL-6. IL-11 and stem cell factor (SCF», as well as the cytokines implicated in decreasing stem cell cycling (transforming. growth factor (3 (TGF-(3) and possibly macrophage inflammatory protem 1a (MIP-la», as the cytokines implicated in decreasing stem cell cycling have pleiotropic effects. These cytokines have effects on cells throughout haemopoietic development as well as on other ~e~elopmentallin~ages:Thus, it appears that most regulators of haemopOletlc stem cell proliferation are not developmentally restricted. In this review. we will describe the haemopoietic regulatory properties of Bai//ierb ClinicalHaematologyVol.S,No.3.July 1992 ISBN (}-7~1628-4

Co . h 703 . pyng t © 1992, by Bailliere Tindall All nghts of reproduction in any form reserved

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two cytokines, TGF-/3 and IL-1, to illustrate how cytokines can act in opposition in order to maintain homeostasis in haemopoietic tissue. A variety of tissues and cell types both express receptors for and produce IL-1 (Dinarello, 1991) and TGF-/3 (Roberts and Sporn, 1990). IL-l is the prototype of the proinflammatory molecule, while TGF-/3 is essentially anti-inflammatory. IL-I is part of a cascade of cytokines that are produced, for example, during microbial invasion or bodily injury, to enhance the host's immune and haemopoietic responses, while TGF-/3 predominantly functions as an inhibitor of these responses. Studies in vivo have demonstrated that both molecules have therapeutic potential. However, chronic overproduction or in vivo administration of these cytokines can have debilitating effects on normal host functions. Thus, regulating the production of these cytokines and/or antagonizing their effects has become therapeutic strategy for various diseases. As might be expected, novel regulatory pathways have been developed by organisms to protect against overproduction of, and chronic response to, these factors. For example, a contra-IL-! (IL-IRa), isolated from the urine of patients with monocytic leukaemia, was found to bind to the IL-l receptor with similar affinity as IL-1, and to prevent IL-1 binding without eliciting a biological response (Carter et ai, 1990; Eisenberg et al,1990). Unique among growth factors, TGF-13 is secreted in a biologically inactive form. In its 'latent' form, TGF-13 is non-covalently complcxed with the amino-terminus of the precursor polypeptide. This complex must be dissociated before it can bind to its specific receptor, adding an additional regulatory step to TGF-13 action. The molecular and cellular biology of both IL-l and TGF-13 have been described in detail in several reviews (Moore, 1989; Massaque, 1990; Roberts and Sporn, 1990; Dinarello, 1991) and therefore will not be discussed except where it pertains to haemopoiesis. The list of cytokines involved in regulating haemopoietic cell development is growing and much work is needed to determine their roles in this process. Thus, this review is not meant to convey the impression that IL-1 and TGF-13 are the only, or even central, players in these events, but rather to serve as a model to illustrate the important role of counteracting cytokines in haemopoietic development. In mammalian tissues, several forms of TGF-13 have been identified and these are identified below as TGF-/31, -/32, -/33 where appropriate.

EFFECT ON GENE EXPRESSION BY IL·! AND TGF·Jl IL-1 induces the production of a variety of cytokines important in haemopoietic cell growth, differentiation and function. It induces the production of mRNA and protein for granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), colonystimulating factor 1 (CSF-1), IL-6, IL-8, tumour necrosis factor a (TNF-a) and TGF-13 in bone marrow derived stromal cells and macrophages. On the other hand, TGF-13 inhibits the increased production of all these factors

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INTERACIlON OF TGF-J3 AND IL-I Table 1. Effects of Ilot and TGF-13on stromal celli macrophage cytokine production. Cytokine production (L-Ia,IL-I13 1L-6 TNF-a TGF-13 G-CSF GM-CSF Arrows indicate an increase ( cytokine production.

IL-l

t t t t t t

TGF-13

t

~ ~

~

1 ~

t ) or decrease ( ~ ) in

Table 2. Effect of IL-I and TGF-13on immunocompetent T-cell cytokine production. Cytokine production IlolRa IL-Ia Ilo2 GM-CSF

11.-4 (loS 1L-6 TGF-13 Arrows indicate an increase ( cytokine production.

IL-t

t t t t t t i

TGF-13

I ~ ~ ~ ~ ~

i t ) or decrease ( ~ ) in

except TGF-J3 (Table 1). In comparison, IL-l production is at first stimulated and then inhibited by TGF-J3 (Wahl et aI, 1987). In T cells, IL-1 amplifies the production of many cytokines (Table 2), while TGF-J3 inhibits the production of most of them (Espevik et aI, 1987; Chantry et aI, 1989). It is interesting that one of the few genes that TGF-J3 activates in T cells is the IL-IRa (Turner et aI, 1991). This suggests that IL-IRa is involved in the ability ofTGF-J3 to suppress IL-l functions . In addition, the effect ofTGF-J3 can be bidirectional. For instance, it has been reported that TGF-J3 can enhance IL-6 production in human peripheral blood monocytes (PBMC) (Turner et aI, 1990) but yet can inhibit IL-l induced production of IL-6 in those same cells (Musso et al, 1990). The mechanism of activation of gene expression by IL-l is in part mediated through the activation of nuclear transcription factors. Two such factors shown to be IL-l inducible are Nuclear Factor (NF)KB (Shirakawa and Mizel, 1989) and Acti~ator Prote~n (A~)-1 (Muegge et aI, 1989). The TGF-J31 promoter has multiple AP-l sites (Kim et aI, 1990) which are active in haemopoietic cells (B.irchenall-~~~erts et aI, 1990): 'Additionally, IL-l can function by prolonging or stabilizing the mRNA half-life of GM-CSF (Griffin et al, 1990) andlorthrough a post-translational mechanism as in the case of TGF-J31 (Bristol et al, 1990). '

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EFFECTS ON T LYMPHOCYTES Using freshly obtained thymocytes, IL-I acts as. co-stimulator of T-cell activation with suboptimal concentrations of antigens or mitogens. IL-! amplifies T-cell proliferation by inducing the gene expression of IL-2 and its receptor. The activation of mature T ceIls is greatly increased when IL-6 is added to IL-I. They act synergistically to increase IL-2 production. Thus, IL-I serves as an augmentation factor and not a true growth factor. Recent studies have shown that TGF-~I and TGF-~2 are equaIly effective in suppressing the proliferation of murine thymocytes in response to IL-I, IL-2, IL-4, IL-6, IL-7, TNF-a and phytohaemagglutinin (PHA). The inhibitory activity is dose dependent over the concentration range 0.4-100 pM and the half-maximal inhibitory concentration for both forms of TGF-~ is approximately 5 pM (Ellingsworth et aI, 1988). Inhibition of IL-l induced thymocyte growth is one of the most sensitive assays for TGF-I3. Furthermore, the suppressive effect of TGF-13 on thymocyte proliferation can be overcome by adding a high concentration of exogenous IL-2, suggesting that TGF-13 inhibits thymocyte proliferation by preventing the IL-I induced production of IL-2. The localized production of TGF-~1 within medullary reticuloepithelial cells suggests a role for TGF-13 in regulating the clonal expansion of the developing T cells. TGF-131 and TGF-132 inhibit mitogen-induced human peripheral blood T cell proliferation. The proliferative response is inhibited (75-90%) in a dose-dependent fashion over the range 10- 12_10- 10 M. Kehrl et al (1986) have shown that TGF-~1 also inhibits IL-2 mRNA expression by mitogenactivated tonsillar T ceIls. Furthermore, these T cells can be induced to express TGF-13 mRNA shortly (2h) after mitogen stimulation. Secreted TGF-I3, however, is not detected in the culture supernatant until 4-5 days after PHA activation, suggesting that TGF-~ may function in an autocrine manner to limit the extent of T-cell clonal expansion (Kehrl et aI, 1986). As with thymocyte proliferation, the inhibitory effect on the growth of activated T cells can be overcome by increasing the concentration of IL-2, suggesting that it is the combined effect of opposing stimulatory and inhibitory signals that determines the growth response of T cells. IN VITRO CYTOKINE-DRIVEN IIAEMOPOIETIC CELL GROWTH

Committed progenitor cell assays In general, two methods are used to study cytokine-mediated haemopoietic cell growth and differentiation in vitro: (1) measurement of cell number and type in suspension culture; and (2) colony formation by different progenitors in semisolid media. A variety of cytokines which act alone or in synergy with other molecules to promote the growth of haemopoietic progenitors has been described (Metcalf, 1989). The cytokines which act directly on maturing haemopoietic progenitors include GM-CSF, G-CSF, CSF-I (macrophage-CSF) and erythroprotein (EPG). all of which induce

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707

terminal maturation of the cell types for which they are named, together with IL-3, which induces the formation of multipotent colonies (CFUGEMM) consisting of granulocytic, megakaryocytic, erythroid and monocytic cells. In contrast, neither IL-1 ncr TGf-B can act alone to promote the growth of any of these progenitors. On the other hand, TGF-~l is a potent inhibitor of IL-3 induced murine bone marrow growth and colony formation but has little or no effect on the growth and differentiation induced by G-CSF, EPO or CSF-1 (Keller et ai, 1988). In addition, while TGF-~l inhibits early erythroid differentiation (CFU-GEMM), which is stimulated by EPa in combination with other factors such as IL·3, TGF·~ has no effect on EPa induced terminal erythroid differentiation. TGF-~ is also a potent inhibitor of cytokine driven megakaryocytopoiesis (Ishibashi et ai, 1987). Also, while TGF-~ inhibits IL·3 induced granulocyte-macrophage colony-forming cell (GM-CFC) colonies, it enhances the growth of GM-CSF induced GM-CFC colonies (Keller et ai, 1991). Similar results were observed with human bone marrow cells, with the exception that the colonies driven by human GM-CSFwhich, unlike mouse GM·CSF, stimulates CFU-GEMM formation, are also inhibited by TGF-~l (Sing et ai, 1988). Furthermore, the action of all cytokines tested, whether they act in synergy or through accessory cells (Broxmeyer et al, 1988), are inhibited by TGF-~ in a dose dependent manner (Ruscetti et aI, 1991). In contrast to previous reports which found TGF-~2 essentially to be inactive in some assays (Ohta et al, 1987; Ottoman and Pelus, 1988), we found that it is almost as effective an inhibitor as TGF-~l, with only a 2-S-fold difference in potency depending on the haemopoietic cells used (Keller et al, 1990a). These differences may be due to the fact that TGF-~2 can be more rapidly inactivated than TGF-~l (Cheifetz et ai, 1991). Recently, it was shown that TGF-~3 is a more potent (lo-Z0-fold) inhibitor of human and mouse haemopoietic cell growth than TGF-rH or TGF-~2 (Jacobsen et al, 1991a,b). These differences in potency (TGF-~3> TGF-rn >TGF·~Z) are also reflected in receptor binding (Massague, 1990) and, unless indicated otherwise, all the data presented in this paper pertain toTGF-IH.

HIGH PROLIFERATIVE POTENTIAL COLONY·FORMING CELLS (HPP.CFC) The slowly cycling murine bone marrow cells that survive a single injection of 5-fluorouracil (5FU) are one of the most primitive haemopoietic cells yet found to grow and differentiate in vitro (Bradley and Hodgson, 1979). These cells, termed high proliferative potential colony-forming cells (HPP-CFC) do not respond to a single growth factor but require at least three of them fa; cell proliferation (Bartelmez et ai, 1989). Both IL-1a and IL-l~ synergize with G-CSF, GM-CSF, CSF-l, IL-3, IL-6 and SCF in the development of HPP colonies and the resultant colony morphology is dependent on the

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cytokines used (McNiece et al, 1990; Moore, 1991). In the mouse, HPPCFC-l colony formation requires the combination of IL-l, CSF-l and IL-3. IL-l can be replaced in this assay by the addition of G-CSF or IL-6. IL-l by itself has no effect on stem cell proliferation or differentiation. Furthermore, since IL-l stimulates the production of IL-6, CSFs and potentially SCF, its actions on early haemopoiesis may be indirect. The growth of all HPP-CFC colonies, regardless of the factors used, is inhibited by TGF·~l in a dose-dependent manner (Keller et al, 1990b). In addition, where IL-l acts to synergize with cytokines to stimulate colony formation, TGF-~ is an inhibitor of that colony formation (J. R. Kelleret al, unpublished data). The same is true for SCF induced colony formation (I. McNiece et al, unpublished data). Bradley et al (1991) recently confirmed this inhibition of HPP colony formation and, more importantly, showed that a 25-fold excess of all, but not one, of the stimulators can overcome the action of TGF-~ . This suggests, as in the case of T-cell prol iferation, that the proliferative status of primitive haemopoietic progenitors is determined by a balance of interacting stimulatory and inhibitory regulators. The actions of several cytok ines, such as G-CSF and GM-CSF, which have growth stimulatory actions on both primitive and more mature human progenitors are affected differentially by TGF-~. For example, TGF-~ inhibits the ability of these cytokines to stimulate growth of primitive progenitors (HPP-CFC, CFU-GEMM and erythroid burst-forming units (BFU-E» but does not affect the more committed progenitors (G-CFC or GM-CFC). Therefore, TGF-~ is a direct selective inhibitor of haernopoietic cell growth that is dependent on the state of differentiation of the target cell rather than on the stimulating cytokine. The effects of TGF-~ and IL-I on haemopoietic progenitor cell growth are summarized in Table 3. Table 3. Effect of TGF-13 on cytokine mediated haemopoietic cell growth. Cytokine

Haemopoietic colony formation Primitive

Mature

Ilol

TGF·13

n-i

TGF-/3

IL-3 IL-6+ IL-3

S S

I I

NE

G-CSF CSF·l GM-CSF

S S S

I I liS

NE NE NE

lINE I NE NE SINE

Primitive colonies include IIPP-CFC, BFU·E, CFUGEMM and CFU·MEG; mature colonies include GM-CFC, G-CFC , CFU-E and CFU·Eos. NE , no effect; I, inhibited, S, stimulated .

DIRECT EFFECTS ON PRIMITIVE HAEMOPOIETIC CELL GROWTH

Since the pleiotropic effects of TGF-~ and IL-l on progenitor cells could potentially be indirect, primitive murine, Thy-f--positive, lineage negative

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(Thy-l + Lin -) bone marrow progenitors (Spangrude et aI, 1988) were isolated and plated in single cell cloning assays. TGF-IH inhibited IL-3 induced growth of Lin-, Thy-l + cells in a dose dependent manner, demonstrating that the effects of TGF-J3 are direct (Keller et al, 1990b). Moreover. if synergistic factors like G-CSF and IL-6 (Ikebuchi et al, 1987; Leary et al, 1988) were added to these cultures, the frequency of responding cells was significantly higher: TGF-J3inhibited this amplified cell growth (J. R. Keller et al, unpublished results). In the same assay, IL-l failed to synergize with IL-3. again suggesting that its effects on haemopoiesis are indirect. Also, using purified CD34+. CD33- human bone marrow cells requiring basic Fibroblast Growth Factor (FGF) , GM-CSF, IL-3 and EPO for optimal colony formation (>75% plating efficiency), TGF-13 at 1 ng/ml could inhibit colony formation, suggesting a direct action of TGF-13 (Sargiacomo et al, 1991). REGULATION OF CYTOKINE RECEPTOR EXPRESSION It is not clear how TGF-J3 and IL-l regulate each other's activities but one possibility is through regulation of the expression of specific cytokine receptors. The specific binding of IL-l to growth factor dependent myeloid progenitor cell lines is inhibited by 50% after a 6 h treatment and by >90% following a 24h incubation with TGF-13 (Dubois et al, 1990a). This effect is also seen on freshly isolated progenitor cells obtained from 5FU-treated mice and is not related to direct competition for the IL-l receptor either by TGF-13 itself or by occupancy and subsequent down-modulation of the receptor by endogenously produced IL-l. Additionally, TGF-13 does not change the level or rate of IL-l receptor internalization (C. M. Dubois, unpublished data). On the other hand, IL-6, G-CSF, IL-3 and GM-CSF were able to up-regulate the number of receptors for IL-1 on haemopoietic bone marrow cells without affecting their affinity (Dubois et aI, 1990b). TGF-13 can also block the ability of IL-3 to up-regulate IL-l receptors. In addition. TGF-J3 inhibits the expression of IL-l receptors on lymphoid cell lines. Interestingly, TGF-13 not only inhibits the ability of IL-1 to stimulate HPP colonies from 5FU-treated marrow cells but also inhibits its stimulation of IL-2 production by lymphoid cells. This suggests that regulation of cytokine receptors is one mechanism by which TGF-13 modulates cellular function. Purified lymphoid and myeloid progenitor cells possess barely detectable levels of TGF-J3 receptors, which increase after cytokine stimulation (Falk et al, 1991a). For example, IL-l and PHA stimulation ofthymocytes greatly enhances the cell surface receptors for TGF-13 within 24 h (Ellingsworth et al, 1988). Similarly. TGF-J3 inhibits the expression of GM-CSF. IL-3 and G-CSF receptors on murine factor dependent myeloid progenitor cell lines previously shown to be ~rowth inhibitab.le byTGF-13 (Jacobsen et al, 1991b). In contrast to the rapid downregulation of IL-l receptors on these cells maximum reduction in the expression of CSF receptor numbers of 65-80%

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is observed only following 72-96 h of TGF-(3 treatment. However, using primary purified murine haemopoietic progenitors, the down-regulation of IL-3 receptors by TGF-(3 is more rapid, with maximum effect being observed within 24h (5. E. W. Jacobsen et al, unpublished data). This TGF-f3 induced trans-dawn-modulation of growth factor receptor expression is prolonged and does not lead to cell death. Moreover, the removal of TGF-f3leads to a recovery of receptor numbers which precedes the resumption of factor mediated cell growth. Taken together, TGF-(3 might function to control immune and haemopoietic cell growth and proliferation by transdown-modulating receptors for stimulatory signals and by inhibiting their induction . Several lines of evidence establish the specificity of this receptor modulation. First, the expression of several different cell surface antigens on haemopoietic cells are not affected by the agents which up-regulate the receptor. Second, two molecules (TNF-a and 'V-interferon) will inhibit the growth of haemopoietic progenitor cell lines without affecting the number or affinity of CSF receptors on the cells (Jacobsen et al, 1991b). Finally, on freshly isolated large granular lymphocytes, the expression of the (3 chain of the IL-2 receptor is not affected by TGF-(3, while on the same cells the expression of the a chain of the IL-2 receptor is markedly downregulated (Ortaldo et al, 1991). Thus, TGF-(3 does not inhibit the surface expression of all cell surface antigens or receptor molecules, even those on the same cells.

LONG-TERM MARROW CULTURE INITIATING CELLS AND BLAST CELL COLONIES: CELL CYCLE ACTIVATION

The available data suggest that TGF-f3 inhibits the proliferation of the pluripotent haemopoietic stem cell (PHSC). Unfortunately, there is no assay for the PHSC other than the ability of transplanted cells to restore haemopoiesis after treatment that results in permanent marrow aplasia (Bertoncello et al, 1988; Jones et ai, 1990). However, a long-term marrow culture (LTMC) system that supports the growth of haemopoietic progenitor cells in vitro for several weeks has been developed (Dexter et al, 1977). The system was first established to support the growth of mouse CFU-S replication. Subsequently it was adapted for growth of human cells (Moore et ai, 1979; Coulombel et ai, 1983). The cells capable of initiating LTMC appear to be very primitive bone marrow cells, since bone marrow cells, highly enriched for day 12 CFU-S, are inadequate (Spooncer et al, 1985). Further separation using rhodamine-123, however, showed that the bright cells included most of the CFU-S but that the rhodamine 'dull' cells alone could initiate and maintain long-term growth in vitro (van der Sluijs et ai, 1990). To maintain the proliferation of primitive progenitors in LTBM cultures, the medium is changed weekly and supplemented with fresh horse serum. Most progenitors enter cell cycle shortly after refeeding. IL-1, which induces

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711

the production of IL-6, G-CSF and GM-CSF in these cultures, can replace the horse serum requirement (Cashman et al, 1990). In addition, TGF-131 can block the ability of IL-1 or horse serum to stimulate progenitor cell cycling and maintain the cells in a non-cycling state. Finally, it has also been shown that neutralizing antibodies to TGF-13 can prolong the cycling of progenitors or enhance the number of cells cycling when added to quiescent LTMC cultures without adding fresh horse serum or IL-1 (Eaves et al, 1991). These results show that TGF-13 is produced and activated by stromal cells in the LTMC and can antagonize the effects of stimulatory signals like IL-t. The most likely candidate for activating 'latent' TGF-13 is the macrophage, which has been shown to be effective with the latent recombinant TGF-131 (Twardzik et aI, 1990). The blast cell colony assay developed by Ogawa and his co-workers allows one to compare the rate of colony formation to determine the effects of cytokines on the recruitment of the colony-forming cells into cell cycle (Ikebuchi et al, 1987; Leary et al, 1988). G-CSF and IL-6 greatly reduce the time necessary for IL-3 to initiate blast cell colony formation of cells from the spleens of 5FU-treated mice. IL-1 is less effective than IL-6, again supporting an indirect mechanism for IL-1, while TGF-13 is able to block the ability of IL-6 to initiate this blast cell colony formation (Kishi et al, 1989).

TGF-13 AND GM-CSF: BIDIRECTIONAL MODULATION OF MYELOPOIESIS IN VITRO Surprisingly, in the presence of murine GM-CSF, TGF-13 promotes a 3-5fold increase in the number, and a substantial increase in the size, of bone marrow GM-CFC colonies (Keller et al, 1991). Morphological examination of the cells present in the colonies shows that the larger size is due to an increase in mature granulocytes. Similarly, in suspension culture, TGF-13 and GM-CSF increases the total number of viable cells, with markedly enhanced neutrophil differentiation along with a concomitant relative decrease in cells of the monocyte-macrophage lineage . Bone marrow progenitors, recovering at 2 and 3 days after treatment with 5FU, respond to GM-CSF plus TGF-I3, while neither cytokine alone has any effect. These progenitor cells, .therefore, see~ to req~ire both signals for growth. In addition, TGF-13 induced a 50% increase 10 the number of GM-CSF receptors on bone marrow cells (Keller et al, 1991). In contrast, TGF·13 inhibits progenitors that respond to growth factors other than GM-CSF, present in 5FU-treated marrow. Single cell cloning assays have proved that both TGF-13 and GM-CSF act directly on single cells to promote growth (Keller et al, 1991). Furthermore, previous ~esults show~d that TGF-13 in the presence of different sources of GM-CSF IS able to stimulate human day-7 but not day-14 GM-CFC colony formation (Ottman and Pelus, 1988; Aglietta et aI, 1989). Jacobsen et al (1991a) showed that the dose ofTGF-13 was critical for this synergistic effec.t on .GM-CSF stimulated human GM-CSF. Thus, TGF-13 can act as a bifunctional regulator of haemopoietic cell growth.

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IN VIVO EFFECTS In vivo injection of IL-1 has profound haemopoietic effects, including production of high serum levels of CSF activity which peaks after 2 h (Vogel et al, 1987), peripheral blood neutrophilia which peaks at 4 h (Stork et ai, 1988), and cycling of bone marrow progenitors (Neta et al, 1987). Despite extensive evidence for the stimulatory effects of IL-l , the mechanisms by which it elicits this response , or with which progenitor cells it interacts, remain to be established. Therefore, we studied the in vivo effects of IL-1 on the expression of IL-l receptors. Both IL-l and G·CSF administration induce the up-regulation of IL-1 receptors on mature haemopoietic cells as well as on a progenitor enriched cell population, with peak effect seen at 12-16h (Dubois et ai, 1991).Furthermore, TGF-13 blocks this process (C.M. Dubois, unpublished results). The existence of two distinct types of IL-1 receptor has been demonstrated by the cloning of two unique cDNAs for IL-1 receptors (Bomsztyk et ai, 1989~ Chizzonite et ai, 1989). The type I IL-lR is expressed on T cells, fibroblasts and epithelial cells, and the type II is on B cells, macrophages and granulocytes. It was reported that pretreatment of mice with anti-type IR antibody blocked the ability of IL-1 to induce IL-IR expression on bone marrow cells without directly binding to those cells (Dubois et al, 1991).This antibody also blocks the ability of stromal elements to produce CSFs in response to IL-l (Neta et al, 1990). These observations suggest that: (1) haemopoietic progenitors possess type II IL-1 receptors; and (2) IL-l mediates the up-regulation of these receptors indirectly through the type I receptors. To determine whether CSFs mediate the effects of Ilo1 in vivo, we showed that: (1) G-CSF but not IL-1 can up-regulate IL-l receptors on bone marrow cells in vitro (Dubois et ai, 1990b); and (2) in vivo injection of G-CSF upregulates the expression of IL-1 receptors on bone marrow cells. Administration of IL-l to mice also results in a rapid elevation of glucocorticoids (GCs). Since in vitro treatment of human monocytes with GCs upregulates IL-l receptor expression (Akahoshi et ai, 1988), it is possible that GCs synergize with CSFs to up-regulate IL-l receptors on haemopoietic cells. Indeed, a partial inhibit ion of the ability of IL-! to up-regulate its receptor on bone marrow cells from adrenalectomized mice was recently observed. Finally, treatment of mice with GCs and CSFs resulted in a synergistic effect on IL-1R expression on bone marrow cells (Dubois et al, 1990c),suggesting that endogenous production ofGC may also playa role in the IL-1 mediated effects on haemopoiesis. The effect of TGF-(3 on haemopoiesis has also been studied in vivo. An approach was developed to administer recombinant TGF-131Iocoregionally to the bone marrow via direct injection into the femoral artery to avoid first pass hepatic clearance (Goey et ai, 1989). Using this route, a single injection of TGF-13 potently inhibited constitutive and IL-3 driven bone marrow growth. This inhibition was also selective in that 100% of CFU-GEMM was inhibited, while only 50% of the more mature committed colony forming cells was affected. This inhibition was both time- and dose-dependent, with the maximum effect being observed at 24 h and at a dose of 5 ug/mouse.

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713

Intraperitoneal injection of TGF-13 proved to be just as effective as local injection (Jansen et aI, 1991). Studies investigating the effect of chronic administration of higher doses of TGF-13 on a variety of haematological parameters were carried out by Carlino and his co-workers (1990). After the subcutaneous daily injection of TGF-13 for 14 days, there was a decrease in mature erythroid cell and platelet numbers, together with an increase in white cell counts in the peripheral blood. Increased granulopoiesis was observed in the spleen and the bone marrow while there was no change in circulating neutrophil counts. The neutrophil has a short half-life and more than 1010 are produced daily. Thus, since neutrophil counts remain normal throughout TGF-13 treatment, in spite of strong inhibition of primitive stem cell proliferation, this suggests that TGF-13 is still able to stimulate neutrophil production. These results correlate well with the in vitro observation that TGF-13 enhances in vitro colony formation in response to CM-CSF (Keller et aI, 1991). A contribution from endogenous G- or GM-CSF, which are known to stimulate neutrophil production by increasing proliferation amplification of the postprogenitor cells (see Chapters3 and 13), cannot be ruled out. Indeed, it was stated earlier that TGF-13induces GM-CSF receptors and recent studies using a sequential administration ofTGF-13 followed by GM-CSF showed an increase in all the granulocyte progenitors in the marrow (Hestdal K et al, unpublished observations). As with a single dose of TGF-I3, the effects of chronic administration of TGF-13 were completely reversible (Carlino et ai, 1990). The in vivo effect of protracted TGF-f3 administration on haemopoietic progenitor stem cells has also been studied by injecting 2.5 j-Lg/mouse intrapcritoneally twice a day (M igdalska et al, 1991). The number and cycling status of the day-12 CFU-S, day-S CFU-S and IL-3 responsive CFU-C were not affected in the first 18 h. However, by day 3, only 50-60% of the day 12 CFU-S were detectable and only 20% of them were in cell cycle. By day 5, none of the cells were in cycle. The prolonged kinetics required to inhibit CFU-S cycling indicates their need first to develop functional TGF-13receptors (Falk et al,1991a). Thirty-six hours after ending TGF-13 treatment on day 5, 130% of the original number of day -12 CFU-S cells were detectable and all the cells were again in cycle , showing the rapid reversibility of the TGF-13 effects on haemopoiesis. As before, there was a modest increase in peripheral white blood cell counts with no adverse effect on neutrophil numbers. Remarkably, most of the effects of TGF-13 on haemopoiesis predicted by in vitro data have been seen in vivo. The role of TGF-f3 in maintaining the slowly cycling status of the PHSC in the marrow remains to be determined; however, it is clearly distinct from other bone marrow inhibitors (Hampson et aI, 1991).

IN VIVO EFFECTS IN COMPROMISED ANIMALS Due 10 its potent stimulatory effects on the bone marrow It-1 was investigated fo! its potential use to protect organisms from d~se/limiting myelosuppresstvc effects that result from bone marrow injury. A single dose

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of IL-1 given 20 h earlier, can protect mice from a lethal dose of'Yirradiation (Neta et al, 1986). It appears likely that the IL-1 effect on radioprotection is indirect. For example, in vivo administration of antibodies to the type I IL-1 receptor that is not expressed on haemopoietic cells can significantly reduce the radioresistance of IL-1 treated mice (Neta et al, 1991). As would be expected from the observations that TGF-J3 down-modulates the IL-1 receptor, stimulates the production of the' IL-1 receptor antagonist and inhibits CSF production by IL-1, administration ofTGF-J3 in vivo enhances the radiosensitivity of mice, presumably by blocking endogenous IL-1 function. IL-1 has also been shown to reduce the severity and extent of neutropenia in 5FU-treated tumour bearing animals (Moore and Warren, 1987; Futami et al, 1990). A 7 day pretreatment with IL-l protects mice against the acute toxicity (LD 90) of 5FU, cyclophosphamide, BCNU and carboplatin but not cisplatin and adriamycin, drugs whose major toxicity is non-haernopoietic (Wiltrout R.H. et al, unpublished results). This pretreatment of mice with IL-1 reduced the nadir of myelosuppression induced by 5FU somewhat but greatly accelerated the regeneration of GM-CFC and peripheral white blood levels. The implications of these findings is that IL-l and other cytokines could be useful for dose intensification of active chemotherapeutic drugs. Futami et al (1990) have demonstrated that this approach can lead to increased therapeutic efficacy in mice.

EFFECT ON LEUKAEMIC CELL GROWTH Acute myeloblastic leukaemia (AML) cells have been shown to produce a variety of cytokines such as IL-1, IL-6, TNF-a, G-CSF and GM-CSF. It has been proposed that IL-1 production by AML blast cells supports autocrine growth in culture by induction of GM-CSF (Griffin et al, 1987; RodriquezCimadevilla et aI, 1990). Among a survey of inhibitory cytokines, TGF-J3 was the only cytokine that produced a clear inhibition of growth (Keranqueven et al, 1990). These data, in addition to our previous report that the growth of chronic myeloid leukaemia (CML) cells (Sing et al, 1988; Aglietta et al, 1989) and most myeloid cell lines are inhibited by TGF-J3 (Keller et al, 1989), demonstrate that many myeloid leukaemic cells are not deficient in responsiveness to TGF-J3.

ROLE OF TGF·1l IN DIFFERENTIATION OF HL·60 MYELOID LEUKAEMIA CELLS Since myeloid leukaemia cells respond to TGF-J3, we investigated whether this response was solely antiproliferative and/or whether their differentiation was affected. Retinoic acid (RA) has been shown to be remarkably effective at inducing differentiation and obtaining remissions in patients with acute promyelocytic leukaemia (Meng-er et al, 1988). Since HL-60 is a pro myelocytic cell line and can undergo granulocytic differentiation with a concomitant decrease in cell growth in response to RA in vitro, we examined

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the role of TGF-J3 in this process. At suboptimal concentrations of RA (t.Ona), which has no effect on either cell growth or differentiation, addition of TGF-J3 markedly inhibits cell growth. This is solely a proliferation response: there is no detectable effect on differentiation (Falk et al, 1991b). RA induces TGF-J3 receptor expression on HL-60 cells in a doseand time-dependent manner with preferential expression of a 53 kDa protein. Interestingly, other inducers of HL-60 differentiation, i.e. PMA and DMSO, also increase TGF-J3 receptor expression. In addition, RA treatment induces increased expression of TGF-IH mRNA, along with increased secretion of active TGF-J3, suggesting that TGF-J3 plays a role in the antiproliferative effects of RA but does not mediate the differentiation. On the other hand, combinations ofTNF-a and TGF-J3 were able to act in synergy to inhibit normal human haemopoietic colony formation (Sing et al, 1989) as well as inhibiting proliferation and stimulating monocytic differentiation of HL-60 and U-937 cells (De Benedetti et ai, 1990). In both cases TNF-a was able to stimulate increased TGF-J3 receptor expression, further suggesting that regulation of functional TGF-J3 receptors plays a role in growth arrest and differentiation of leukaemic cell lines. INHIBITORS AND LYMPHOMA CELL GROWTH Since escape from inhibitory regulators such as TGF-J3 could playa role in the growth of neoplastic cells, the effects of TGF-J3 on lymphoid leukaemic cells has also been examined. TGF-J3 exerts profound inhibitory effects on the growth of both normal Band T lymphocytes. In contrast, a number of lymphoid cell lines are insensitive to the antiproliferative effects ofTGF-J31 and TGF-J32 (Sing et ai, 1990). In particular, binding and crosslinking with radio iodinated TGF-J31 demonstrated the absence or low expression of all three TGF-J3 receptor species on three B-cell tumour lines, while T-cell, non-T and non-B tumours expressed large numbers of receptors. Treatment of the B-cell lines with PMA induced the expression of TGF-J3 receptors and inhibited proliferation in all three lines in a dose- and time-dependent manner. The cell lines constitutively produced TGF-J3 mRNA and low levels of latent TGF-J3. However, PMA also induced increased expression of TGF-J3 mRNA and, more importantly, the release of active TGF-J3. A neutralizing antibody to TGF-J3 was able to reverse the PMA-induced growth inhibition, and addition of exogenous TGF-J3 reversed the effects of the neutralizing antibody. Thus, TGF-J3 can inhibit human lymphoma cell growth in vitro through an autocrine mechanism. Some lymphoma cells appear to escape from TGF-J3 suppression by failing to express functional TGF-J3 receptors and/or failing to secrete active TGF-J3. However, recent studies have shown that Epstein-Barr virus-transformed B cells and Burkitt's lymphoma cells can be either sensitive or insensitive to TGF-J3, without regard for the presence or absence of TGF-receptors on the cell surface (Altiok et al, 1991). In addition, all human T-cell leukaemia virus (HTLV)-I transformed T cells tested are able to bind TGF-J3 but they cannot be inhibited by it. There are clearly several ways to escape regulation by TGF-J3.

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SUMMARY

The polypeptide cytokines, IL-I and TGF-~ affect nearly every tissue and cell type in the body. IL-I is the prototype of the proinflammatory molecule while TGF-~ is essentially anti-inflammatory. IL-I is part of the cascade of cytokines that are produced during microbial invasion or bodily injury and enhance a variety of host responses, particularly in the immunological and haemopoietic systems, while TGF-~ acts as an inhibitor of these responses. A.t several levels, IL-I and TGF-~ act in opposition to one another. IL-I stimulates the expression of many genes in lymphoid and marrow stromal cells that stimulate haemopoietic cell growth and differentiation, while TGF-~ inhibits these IL-I mediated effects. Also, TGF-~ stimulates secretion ofthe IL-IRa. In addition, IL-I induces the cell surface expression of cytokine receptors on lymphoid and haemopoietic cells, while TGF-~ dramatically inhibits the cell surface expression of these receptors, including the IL-1 receptor. Finally, IL-1 augments lymphoid and haemopoietic cell growth and TGF-~ potently inhibits this proliferation. The interactions of these cytokines serve to illustrate that the net balance of stimulatory and inhibitory signals determines the fate of a given cell which may be responsible for regulating homeostatic cell growth (Figure 1). Thus, the regulation of cytokine production and/or antagonism of their effects continues to be a therapeutic goal in the treatment of many diseases.

Figure 1. A schematic representation of the cooperation between stimulatory (black) and inhibitory (white) cytokines in the control of haemopoiesis.

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Acknowledgement This project has been funded at least in part with Federal funds from the Department of Health and Human Services under contract number NOI-CO-74102. The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does the mention of trademarks, commercial products or organizations imply endorsement by the US Government.

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