Cytokines at the research-clinical interface: potential applications

Blood Reviews (1996) 10; 189-200 0 1996 Pearson Professional Ltd

State of the art

Cytokines at the research-clinical potential applications

interface:

T. L. Holyoake Developments in the characterization of growth factors and the recognition of their potential for clinical use has advanced through a number of stages.The development of clonogenic haemopoietic colony assaysin the 1960sled to the discovery of colony-stimulating activity in the conditioned medium produced by certain cell lines. This activity was then purified and the colony-stimulating factors were identified. With rapid progressin molecular biology techniquesin the 1980s many further growth factors were cloned and produced on an industrial scale.Although erythropoietin, interferons, G-CSF, GM-CSF and IL-2 were all introduced into clinical practice as single agents, cytokines have more recently beeninvestigated for use either in combination, or sequentially. Clinical trials are currently in progressto examine the optimum combinations and timing of administration. Current clinical applications include optimization of methods for mobilization of peripheral blood progenitor cells and amelioration of cytopenias following chemotherapy and bone-marrow transplantation. In the future, cytokines will be employed to expand stem and progenitor cells ex vivo, to improve gene transduction strategies,possibly to protect the gastrointestinal epithelium and as immunomodulators, both in vivo and in vitro. This review will focus on recently characterized growth factors including c-kit ligand/stem cell factor, flt3 ligand, c-mpl ligand/thrombopoietin and interleukins- 11,4, 7, 10, 12 and 13. array of CSFs, interleukins and other cytokines has been clonedand producedon an industrialscale.

BACKGROUND

In the 1960sgroupsin Israeland Australia developed in vitro clonogenic assays for the growth of normal murine bone marrow cells.1-3 These culture systems were then extended to the cloning of human myeloid cells and to erythrocytes, B cells and T cells. Such assays facilitated the discovery of a family of cytokines or colony-stimulating factors (CSFs) which have been found to regulate cell viability, proliferation and differentiation. The first CSFs to be discovered included M-CSF, G-CSF, GM-CSF and multi-CSF, later renamed IL-3. However, over recent years, an

CYTOKINE

NOMENCLATURE

The nomenclature used for the many growth factors may initially seem complex. Historically, the early CSFs were named according to the type of colony formation promoted by the purified protein, e.g. G-CSF, GM-CSF, M-CSF and multi-CSF (IL-3); later the numbered interleukins (l-17) were identified by cloning and named in chronological sequence; and, finally, in certain instances the receptor (e.g. c-kit, flt3, c-mpl) was identified before the growth factor which hence became known as c-kit ligand, flt3 ligand and c-mpl ligand. Following commercial development, c-kit ligand is now known as stem cell factor (SCF) and

Tessa L. Holyoake Clinical Research Fellow and Senior Registrar in Haematology, Department of Haematology, Glasgow Royal Infirmary, 84 Castle Street, Glasgow G4 OSF, UK. Tel: (f 44) 0141 2114000, page 1747 or 0141 211 4672 (secretary); Fax: 0141 552 8196.

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IL-10 was identified by its ability to inhibit cytokine synthesis in lymphoid cells.&* Both murine and human IL-10 receptors are structurally related to interferon receptors9

c-mpl ligand as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF). The function of cytokines is not restricted to the haemopoietic system and these factors have important roles in defence against infection, wound-healing, angiogenesis and inflammation.

CYTOKINE CYTOKINE

RECEPTOR

FAMILIES

Many cytokines tend to share a range of biological effects. For example, leukaemia inhibitory factor (LIF), IL-6, IL-l 1 and oncostatin M (OSM) show similar activities in that they promote thrombopoiesis, stimulate the production of acute-phase reactants by hepatocytes, alter neuronal signalling and suppress lipid transport.2 This apparent redundancy is likely to reflect the fact that many cytokines share receptor subunits (see Table 1). The receptors for M-CSF, SCF and flt3-ligand are protein tyrosine kinase-containing receptors which show structural similarity to the platelet-derived growth factor (PDGF) receptor.3 The receptors for many growth factors are members of a cytokine receptor superfamily. This family is characterized by conserved elements in the extracellular domain, suggesting that these receptors are derived from a common ancestral receptor.2,4 These receptors each have a specific a-chain but, whereas GM-CSF, IL-3 and IL-5 share the same P-chain, IL-6, LIF, OSM and IL-l 1 all share another common P-chain - the gp130 molecule. Since the P-chain appears dominant in intracellular signalling, this may explain the similarities of action of GM-CSF, IL-3 and IL-5 and of LIF, IL-6, OSM and IL-l 1. IL-2, 4, 7, 9 and 1.5 form a group of soluble factors that signal via a common receptor component, the yc receptor. Each separate ligand interaction also requires binding to at least one other receptor subunit to specify individuality. IL-4 and IL-13 are two closely related proteins produced by activated T cells which share biological activities. Although the cloned IL-4 receptor does not bind IL-13, it appears that the IL-4 and IL-13 receptors share a common subunit which is important for signal transduction.5 Unlike most other cytokines,

Table Receptor

1 Cytokine tyrosine

receptor

The question of cytokine redundancy has, in part, been answered by the study of animals in which the gene in question has been deleted or functionally inactivated (knockouts).To date, cytokine knockouts of G-CSF, GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-S, IL-lo, LIF, TGF-P and others, yet to be published formally, have been engineered.‘O In addition, mice with a genetic defect causing osteopetrosis (oplop) have been shown to have inactivation of the M-CSF gene” and both Steel (Sl) and white spotting (W) anaemic mice have now been shown to be genetically deficient in SCF and c-kit (its receptor) respectively. I2 The availability of gene knockout animals and mutations occurring spontaneously in vivo, have allowed investigators to learn more about the role of individual cytokines. For example, G-CSF knockouts suffer severe neutropenia but some normal mature neutrophils are still present, suggesting that other cytokines are able to partially compensate for the lack of G-CSF.13 GM-CSF knockouts show pulmonary pathology, but, surprisingly, have apparently normal haemopoiesis.14 c-mpl is a proto-oncogene and a member of the cytokine receptor superfamily.15 The ligand to c-mpl, (TPO), was recently cloned and found to have potent effects on megakaryocytopoiesis.16 c-mpl knockouts have reduced platelet numbers and increased serum TPO levels, but are still able to produce some functional platelets in the absence of signalling via c-mpl. Likewise, op/op M-CSF deficient mice exhibit a major deficiency in macrophagederived osteoclasts and partial deficiency of other macrophage populations which are relieved by injection of M-CSF. However, since these mice are not completely devoid of macrophages, other cytokines must be partly able to compensate for the deficiency.

families

kinase

PDGF,

Cytokine receptor superfamily: non gp130, common-p subunit Cytokine Cytokine common

REDUNDANCY

IL-3,

receptor receptor y chain

superfamily: superfamily:

gp130

Cytokine receptor Interferon-like

superfamily:

other

SCF, M-CSF, GM-CSF,

IL-6,

IL-1 1, LIF,

OSM

IL-2,

IL-4,

IL-9,

TPO IL-10

IL-7,

Flt3

ligand

IL-5 (IL-12) IL-15,

(IL-13)

Cytokines

Sl and W mice show similar phenotypes including severe anaemia and deficiency of very primitive haemopoietic stem cells.

CLINICAL

APPLICATIONS

OF CYTOKINES

It is now five years since G-CSF and GM-CSF were licensed for clinical use in vivo. These cytokines exert powerful effects on haemopoiesis and are effective in a number of clinical areas but are relatively expensive. In recent years, both a European and a US expert panel have established consensus guidelines for the clinical use of growth factors. A remarkable degree of agreement can be found between results of both working parties.17Js There are several more recently identified haemopoietic growth factors currently in clinical trials and others which have shown potential for clinical development. This review will focus on recently characterized growth factors including c-kit ligand/SCF, flt3 ligand, c-mpl ligand/TPO and Interleukins-1 1,4, 7, 10, 12 and 13.

STEM CELL FACTOR The cloning of SCF (otherwise known as c-kit ligand, Steel factor, mast cell growth factor) provided a major advance to the understanding of cytokine interactions which controlled the proliferation of early haemopoietic stem cells. SCF, first identified in 1990,19-‘5 is the ligand for the receptor encoded by the c-kit protooncogene.3 In mice, mutations may either effect the locus of c-kit (White Spotting W locus) or its ligand (Steel Sl locus), resulting in variable defects in pigmentation, fertility and haemopoiesis (reviewed in26). The gene for SCF is found on chromosome 12q22-q24. In humans, the c-kit receptor is expressed on several tissues including melanocytes, haemopoietic progenitor cells and mast cells. SCF exists in both soluble and membrane-bound forms. Sl mutant mice synthesize soluble SCF, which is active in vitro, but fail to make the membrane-bound form. Since these mice exhibit an obvious phenotype, this suggests that membrane-bound SCF has a critical role in stem cell-stromal interactions in the intact organism that is not duplicated by the soluble molecule. The fact that defects in the production or action of SCF lead to bone-marrow failure in mice, suggests that this cytokine is likely to play a major physiological role in stem cell development and in haemopoiesis. As predicted, SCF has been shown to be a potent stimulator and regulator of early events in haemopoiesis.27,28 SCF may shorten the dormant phase of stem cells 29 and enhances survival of highly

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1

enriched murine progenitor cells under serum-free conditions.30 To date, no convincing evidence has yet been published, indicating that SCF promotes selfrenewal of HSC. Acting alone, SCF has little proliferative activity. However it synergizes with a number of cytokines to stimulate growth of primitive and lineagecommitted haemopoietic progenitor cells in vitro.j1,32 In addition to potent effects on granulocyte and microphage precursors, SCF has been shown to exert effects on erythropoiesis,33s34 mast cell generation,2’,23 T-and B-cell lymphopoiesis,26 and megakaryopoiesis? In pre-clinical studies, SCF used alone, in murine, canine and primate models, induced neutropllilia,36 increased stem cell numbers in bone marrow and peripheral blood,34,35 provided a degree of radioprotection37x38and reduced the duration of neutropenia and thrombocytopenia in Rhesus monkeys following high-dose chemotherapy. G-CSF, combined with SCF, has promoted a greater and more sustained mobilization of PBPC,39A1 produced more rapid engraftment of those mobilized cells and reduced the cell dose required for rescue from high-dose chemotherapy. 42 The major adverse effects observed appeared to be caused by reversible hyperplasia of mast cells. The results of pre-clinical studies therefore suggested that SCF might have therapeutic promise when administered in combination with other growth factors such as G-CSF, particularly for the mobilization of PBPC. Mast cell-mediated effects were clearly of concern and it was anticipated that this might cause dose-limiting toxicity in clinical trials. Phase 1 clinical trials of SCF administered alone to patients with lung or breast cancer showed an apparent acceleration of leukocyte and platelet recovery following chemotherapy. Patients suffered skin reactions at the injection site, urticaria and respiratory symptoms which were attributed to mast-cell activation and degranulation. It was therefore suggested that SCF was administered in low doses (20nglkglday) and with antihistamine and H2 receptor antagonist cover (reviewed in”‘). Three subsequent phase I/II trials have focused on the use of SCF in combination with G-CSF for mobilization of PBPC. Data from more than 200 patients with breast cancer or lymphoma confirmed that SCF was well tolerated if patients with a history of atopy were excluded and others were premeditated with antihistamine and H2 receptor antagonists. SCF increased the number of CD34’ progenitor cells that could be harvested in comparison to G-CSF alone even in patients who were heavily pretreated with chemotherapy.43 These trials have not examined the role of SCF for mobilization of PBPC following chemotherapy and it is possible that PBPC mobilization by chemotherapy plus G-CSF may not be further enhanced by SCE Phase III multi-institutional

192 Blood Reviews clinical trials of SCF are currently under way to document the effect of SCF in decreasing leukapheresis requirements. Anticipated future clinical applications of SCF include the ex vivo expansion and gene manipulation of haemopoietic stem and progenitor cells and the treatment of bone-marrow failure states. A clinical trial of SCF in patients with aplastic anaemia is in progress but has not yet been reported.

FLT3 LIGAND The flt3 receptor was isolated independently by two groups in 1991 44-46and was found to be a member of the same family of tyrosine kinase receptors as c-kit, c-fms and the PDGF A and B receptors. The expression of flt3 is relatively restricted. It is found on pre-B cell lines and CD34’ haemopoietic cells, but not on erythroid progenitors nor on mast cells.47b48The flt3 receptor was used by two groups to successfully isolate and clone flt3 ligand in 1993/94.49Jo The gene for flt3 ligand is found on human chromosome 19q13.3.48 Trisomy 19 has been reported to be strongly associated with myeloid malignancies.51 The flt3 receptor gene has been mapped to chromosome 13q12 in humans in a region that is deleted in some patients with myeloproliferative diseases.44,48 In vitro flt3 ligand has weak proliferative effects on primitive haemopoietic cells, and synergizes with IL-3, IL-7, SCF, GM-CSF, PIXY 321 and other growth factors, but not with erythropoietin.48,49 In contrast to SCF, flt3 ligand has no effect on the growth of either erythroid or mast cells.52 Since the deleterious effects of SCF in early clinical trials were thought to be related to mast-cell activation, the lack of activity on mast cells should prove to be a major advantage for clinical development of At3 ligand. Flt3 ligand offers the potential for therapeutic use in a number of clinical areas. This cytokine may promote generation of committed progenitor cells more rapidly from the stem-cell pool, hence producing G-CSF and GM-CSF-responsive target cells following chemotherapy and/or bone marrow transplantation. 53 For patients with limited bone-marrow reserve it is possible that flt3 ligand may expand immature stem cell subsets prior to mobilization with either G-CSF or GM-CSE4* In ex vivo expansion studies, flt3 ligand appears to be unique in its ability to induce expansion of long-term culture-initiating cells to around 30 fold over a lo-day culture period in the absence of a supporting bone marrow stromal layer.54 Finally, for gene therapy applications, flt3 ligand may induce cycling of cells which were previously considered to be growth factor-unresponsive.55

A number of pre-clinical studies of flt3 ligand were reported at the 1995 meeting of the American Society of Hematology. In a non-human primate model, flt3 ligand expanded and mobilized early haemopoietic cells, induced cycling of primitive cells and increased bone-marrow cellularity. Treated animals developed detectable lymphadenopathy and splenomegaly.56 In mice treated with the combination of G-CSF and flt3 ligand, colony-forming cells and colony-forming units-spleen (CFU-S) in peripheral blood, bone marrow and spleen were greatly increased compared with treatment with G-CSF alone, e.g. peripheral blood CFU-S increased 18 fold over baseline with G-CSF and 384 over baseline with G-CSF plus flt3 ligand.57 In mice treated with retroviral vector-mediated gene transfer to overexpress murine flt3 ligand, peripheral blood lymphocytes, neutrophils and monocytes were increased 4-10 fold, whilst platelets were unchanged. Primitive Sea-1’ c-kit+ cells in bone marrow were increased 5-7 fold, early B lymphopoiesis was stimulated and there was evidence of extramedullary haemopoiesis and fibrosis in the spleen. No effects outwith the haemopoietic system were observed.58 Finally, Dao et al reported that the addition of flt3 ligand to the combination of IL-3, IL-6 and SCF allowed viability of human haemopoietic stem cells to be maintained for 72 hours ex vivo and that efficient gene transduction occurred even in the absence of supporting stroma. Transduction and survival of very primitive human haemopoietic cells was then determined by growth in immune-deficient mice for eight months. LN provirus was detected in the marrow of four of nine mice that received human cells transduced in suspension culture with flt3 ligand, confirming for the first time that long-lived haemopoietic progenitor cells may be successfully transduced in vitro in the absence of stroma. These preliminary studies confirm that flt3 ligand is a powerful haemopoietic ‘stem cell factor’ that is likely to find a number of important therapeutic roles in the near future. Flt3 ligand differs from SCF in that it appears to trigger proliferation of very early myeloid stem cells with little effect on progenitors committed to either erythroid or megakaryocytic lineage and no effects outwith haemopoiesis. These features suggest that, in clinical trials, flt3 ligand should prove less toxic than SCF.

C-MF’L LIGANDlTHROMBOPOIETIN Thrombocytopenia is less common than neutropenia following standard chemotherapy protocols, but is a major cause of morbidity following bone-marrow or

Cytokines

PBPC transplantation and intensive regimens for acute leukaemia. Over recent years, there has been an ever-increasing use of platelet transfusions which, although generally efficacious, have associated risks of transmitting blood-borne disease and of alloimmunization. Therefore, there is a clinical requirement for a drug that will reliably alleviate the thrombocytopenia associated with cancer therapy. In 1992, Vigon and colleagues isolated c-mpl, the cellular homologue of v-mpl previously recognized to be the transforming oncogene of the murine myeloproliferative leukaemia virusbo Characterization of the proto-oncogene c-mpl revealed structural homology with the haemopoietic cytokine receptor family, i.e. c-mpl appeared to be an ‘orphan’ cytokine receptor. This major advance soon led to the cloning, by several groups using different strategies, of c-mpl ligand, otherwise named TPO or MGDF.16 The gene for human c-mpl ligand is located on chromosome 3q27. Knockout, transgenic and gene transfer experiments have confirmed the importance and selectivity of c-mpl ligand in the regulation of megakaryocytopoiesis. 12,60-62c-mpl knockout animals have been shown to have low numbers of platelets in peripheral blood, reduced megakaryocytes in spleen and bone marrow and raised thrombopoietin levels. Likewise, thrombopoietin knockout animals have an 80% reduction in both platelets and megakaryocytes. In vitro thrombopoietin induces cell-surface expression of glycoproteins IIbiIIIa and Ib, both on normal cells and on megakaryocyte cell lines, and induces proliferation and maturation of megakaryocyte progenitors.61m63In vivo experiments in normal mice have demonstrated that administration of c-mpl ligand is associated with a 4-fold increase in circulating platelets and a 20-fold increase in bone-marrow megakaryocyte precursors but with little effect on either the erythroid or granulocytic lineage.64 c-mpl ligand may also accelerate platelet recovery, reduce platelet nadirs and improve mortality in severely myelosuppressed mice treated with the combination of irradiation and carboplatin6j Primate studies have confirmed that c-mpl ligand is a lineage-specific cytokine capable of inducing an increase in megakaryocytopoiesis. In rhesus monkeys, and more recently babocms, MGDF produced a dose-dependent increase in platelet counts to 7 fold over baseline.66,67 Studies in normal animals have shown that platelet counts rise following an initial lag phase of several days and take approximately two weeks to return to baseline. In non-human primates given sub-lethal irradiation MGDF, either alone or in combination with G-CSF, has been shown to accelerate platelet recovery. Animals given both MGDF and G-CSF had a shorter duration of neutropenia than those given G-CSF alone suggesting that MGDF and

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G-CSF may safely be combined for the treatment of myelosuppression (reviewed i@). A number of studies have assessed the function of platelets produced in response to c-mpl ligand. In summary, these studies seem to indicate that despite in vitro evidence of sensitization, platelets produced in this manner function appropriately and should not expose patients to undue risk of thromboembolism, particularly if platelet counts are not allowed to rise to excessive levels.h7 Few clinical studies involving c-mpl ligand have been reported to date. Thrombocytopenic patients have been shown to demonstrate an inverse relationship between platelet counts and c-mpl ligand levels consistent with the concept that c-mpl ligand is important in the recovery from thrombocytopenia.68 The first clinical trials of MGDF started over a year ago, The early reports indicate a dose-dependent increase in platelet counts in patients with advanced cancer given MGDF prior to chemotherapy. Crucially, no toxicity has, as yet, been attributed to the drug.69 Several pleiotropic haemopoietic growth factors are currently in clinical trials for the treatment of thrombocytopenia. These include IL-6 IL-3, IL-1 1, GM-CSF and PIXY 321. The effects of these cytokines appear to be modest at most and, with the exception of IL-1 1, their side-effects are likely to limit their clinical application. The major advantage of c-mpl ligand over these growth factors is its lineage specificity which, in early clinical trials, has meant little or no associated toxicity. INTERLEUKIN-11

IL-1 1 was first cloned from a primate bone-marrowderived stromal cell line, PU-34, as a factor which stimulated proliferation of an IL-6-dependent murine plasmacytoma cell line.70 A second group, working separately, cloned a novel adipogenesis inhibitory factor from the human bone marrow derived cell line KM-102, which they later found to be identical to IL-11 (reviewed in 71). Both the murine and human IL-l 1 receptors have recently been cloned and show 82% sequence homology. 72.73IL-l 1 belongs to the family of cytokines that use the gp 130 transducing subunit in their high-affinity receptors. The gene for the ligand is found on human chromosome 19q13. IL-1 1 is a multifunctional cytokine with a range of biological activities that resemble those of IL-6. These effects include stimulation of T-cell-dependent B-cell immunoglobulin secretion,74 synergism with other cytokines to stimulate early multipotential progenitor cells7j and those committed to the erythroid7(j and megakaryocyte lineage, 7o the ability to shorten the dormant period of stem cells,77induction of secretion of acute phase proteins in the liver and the inhibition of adipose conversion in the bone marrow.‘r

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Blood Reviews

IL-l 1 is active in a variety of animal models including rodents, rabbits, dogs and non-human primates. In vivo, in normal rodents, IL-11 stimulates a marked increase in the numbers of bone-marrow megakaryocytes and circulating platelets.78 For mice given IL-11 following BMT, neutrophil and platelet recovery is enhanced, and this is accompanied by increased numbers of progenitors in the bone marrow and spleen. Furthermore, IL-11 enhances antigen-specific responses after cytoablative therapy. Haemopoietic recovery, following administration of cytotoxic agents, is also accelerated by IL-1 1 administration and platelet nadirs are less severe.78 More recently, IL-11 has been shown to protect clonogenic stem cells in murine gastrointestinal mucosa from the effects of radiation and chemotherapy.7g-81 In these studies, IL-l 1 appeared to promote maintenance of the structural integrity of the intestinal mucosa, thereby preventing sepsis. Since IL-l 1 reduces levels of TNF-a, IFN-y, IL-lb, IL-12 p40 chain and nitric oxide, it has been postulated that these antiinflammatory properties may account for the results. A clinical programme to investigate the use of IL11 in patients with chemotherapy-induced thrombocytopenia began in 1992. In clinical studies, IL-l 1 stimulated platelet production and maturation, increased plasma von Willebrand factor and fibrinogen, and reduced thrombocytopenia after high-dose chemotherapy (reviewed in**). It was well tolerated at lo-50 pg/kg with patients developing only mild reversible anaemia, but at doses greater than 75 yglkg it induced oedema, fatigue and myalgia. When IL-11 was combined with G-CSF, after high-dose chemotherapy with autologous rescue, both neutrophil and platelet recovery were accelerated with reduced need for platelet transfusions. In these studies, no increase in platelet reactivity was observed after in vitro exposure to IL-l 1, in contrast to the increase in spontaneous and triggered aggregation observed following either IL-6 or TPO exposure.** Phase III clinical trials are currently under way. In summary, therefore, IL- 11 has been shown to have profound effects on haemopoiesis by stimulating early stem cells and megakaryocytic progenitors. A randomized, placebo-controlled study has demonstrated that IL-l 1 can reduce the need for platelet transfusions and is well tolerated. Its additional, potentially beneficial, effects on the gastrointestinal epitheliums suggest that IL-l 1 should prove a valuable tool in cancer therapy.

IMMUNOMODULATORY

CYTOKINES

In 1986, Mosmann et al discovered that murine helper CD4’ T cells may be divided into two subsets based on

Table

2 The ThllTh2

paradigm

T-cell subset Thl

T-cell response Cell mediated

Th2

Humoral

Cytokines elaborated IL-2, IFN-?I, TNF-cx, TGF-P IL-4, IL-5, IL-6, IL-IO, IL-13. TNF-a

differences in cytokine production on activations3. T helper 1 (Thl) cells secrete IL-2, IFN-y, TNF-a and TGF-P, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-lo, IL-13 and TNF-a. Naive ThO cells, both CD4’ and CD8+, may be driven to differentiate into type 1 or type-Zlike populations by stimulation via the T cell receptor and exposure to certain key cytokines. IL-12, in the absence of IL-4, drives a Thl response and IL-4 drives a Th2 response. The effects of IL-4 in this respect are dominant over IL-12. It is now widely recognized that the human immune response is similarly polarized into type 1 and type 2 responses. In general, type 1 responses are associated with cell-mediated immune processes and type 2 responses are associated with humoral responses and anti-inflammatory processes (reviewed ing4) (see Table 2).

INTERLEUKIN-4,13

AND 10

IL-4 was first described and characterized by cDNA cloning in 1986 as a growth factor that was produced by activated mouse Th2 cells.85 IL-4 has since been shown to have important immunoregulatory functions. IL-13 was first fully described in 1989 as a protein predicted to be encoded by an mRNA specifically produced by activated Th2 cells.86 Although IL-4 and IL-13 have distinct receptors, these are thought to share a common subunit. This may explain why these cytokines share some biological effects. However, there are certain cell types, including T cells, which proliferate in response to IL-4 but fail to respond to IL-13. This observation suggests that T cells may lack functional IL-13 receptors. In general, the biological functions of IL-13 are more restricted than those of IL-4. Both IL-4 and IL-13 act on monocytes and macrophages to change morphology, regulate surface antigen expression and to inhibit antibody-dependent cellular cytotoxicty IL-13, in common with IL-4, has potential anti-inflammatory activity as judged by its ability to suppress the production of pro-inflammatory cytokines. IL-l 3 may also suppress the cytotoxic functions of monocytes and macrophages, including their capacity to kill intracellular parasites (reviewed in5). Like IL-4, IL-13 has been shown to inhibit human

Cvtokines at the research-clinical interface

immunodeiiciency virus (HIV) replication in monocytes.87 IL-13 and IL-4 are both able to downregulate IL-12 and IFN-a production and hence to favour development of a Th2 response. IL-4 and IL-13 have similar activities on B cells but generally IL-4 is more potent. Both IL-4 and IL-13 may also induce IgG4 and IgE synthesis and direct IgE-isotype switching. For therapeutic use, it is likely that blockade of both IL-4 and IL-13 would, therefore, be required to block synthesis of IgE in atopic individuals.88 Although IL-4-deficient mice show normal lymphocyte development and expansion, Th2 responses are blocked.89 In addition, IL-4-deficient animals do not mount an allergic pulmonary eosinophilia in response to specific allergens. 9oTherefore, although IL-4 plays an important role during immune responses, it appears to have no crucial role in lymphopoiesis. IL-10 is a pleiotropic cytokine exerting effects on a diverse range of cell types. It was initially discovered as an activity produced by murine Th2 cells which was able to suppress cytokine production by Thl cells.6 IL-10 is also produced by B cells, monocytes, macrophages and keratinocytes and inhibits synthesis of the proinflammatory cytokines, IL-l, IL-6, IL-8, IL-12, TNF-a, GM-CSF and others.9’ IL-10 is a potent suppressor of macrophage activation and downregulates the constitutive and IFN-a-induced expression of class 11 major histocompatibility complex (MHC) antigens (HLA-DR/DP and DQ) on monocytes.92 However, IL-105 effects are not all inhibitory. It enhances the viability of murine B cells and promotes proliferation and differentiation of human B cells into plasma cells. IL-2 and IL-10 synergize to stimulate normal B cells to secrete immunoglobulins (reviewed in93). IL-lo-deficient knockout mice show normal lymand antibody responses. phocyte development However, they suffer from chronic enterocolitis, suggesting an essential role for IL-10 in immunoregulation of the gastrointestinal tract.94 A number of further studies suggest that IL-10 may be a new therapeutic agent for the treatment of inflammatory bowel disease.9j It is widely recognized that overproduction of cytokines contributes to many of the pathological changes observed in Gram-negative sepsis. TNF-a is thought to be most important in this respect. In a preclinical murine model, a single injection of IL-10 reproducibly protected mice from a lethal injection of endotoxin and this effect was reversed by neutralizing IL-10 antibodies.95 This and other studies implicate IL- 10 as a candidate for the treatment of systemic and local sequelae of septic shock. In the myeloid malignancies, autonomous release of cytokines may play an important role in disease progression which could, perhaps, be inhibited by IL-lo.

195

In clinical trials, therefore, IL-10 may be expected to be useful as a modulator of components of inflammation in diseases such as inflammatory bowel disease, acute pancreatitis, sepsis, arthritis, rejection of transplanted organs and graft-versus-host disease following bone-marrow transplantation. IL- 10 has now been given to normal volunteers in at least two studies.91s96 Acceptable serum levels were achieved with minimal toxicity and effects on T cells and cytokine production were demonstrable. Currently IL- 10 is being evaluated in conditions including Crohn’s disease, rheumatoid arthritis and transplant rejection.

INTERLEUKIN-7

The stromal-derived cytokine IL-7 was originally identified as a growth factor for B-lymphocyte precursors. However, more recently, it has been recognized that IL-7 also affects the activity of T and natural killer (NK) cells.97,98In a recent study, IL-7 was shown to enhance the expression of IFN-y in activated T cells. This effect was shown to be independent of IL-12 secretion and less potent than IFN-y induction observed with IL-12.99 Therefore, IL-7 may be considered as an intermediate cytokine in determining a Thl response. In addition to effects on B and T cells, haemopoietic stem and progenitor cells are responsive to IL-7, which induces mobilization of stem cells into the peripheral blood. loo~lolMobilization of immature progenitors is further increased if IL-7 is administered in combination with G-CSF.98 These pleiotropic effects suggest several potential avenues for clinical development of IL-7, including the prevention and/or treatment of infectious disease and cancer and stemcell mobilization for transplantation. Recent studies have used three strategies for examining the role of IL-7 in lymphopoiesis. These include the generation of IL-7 transgenic mice,98 the administration of IL-7 or IL-7 receptor-neutralizing antibodies *02,103or the production of IL-7 or IL-7 receptor knockout animals.104J05The results of these studies confirm the importance of IL-7 in T-cell and B-cell development and the apparent absence of other cytokines which may compensate for the lack of IL-7. On the other hand, IL-7-deficient mice show no abnormalities of either myeloid cells or NK cells. Pre-clinical studies in mice have shown that IL-7 administration following cytotoxic drugs or irradiation accelerates Tand B-cell regeneration, suggesting that IL-7 may be clinically useful in stimulating lymphoid regeneration following bone-marrow transplantation, particularly in the allogeneic setting. 98 A number of studies illustrate the importance of IL-7 in immune responses in skin and gut. Furthermore, IL-7 in combination with

196 Blood Reviews IFN-y and IL-2 may be of value in expanding antigen-specific T cells for use in adoptive immunotherapy and, in combination with IL- 12, may be useful for augmenting T cell responses in vivo (reviewed in98). A number of pre-clinical studies have demonstrated that IL-7 can induce or modulate anti-tumour responses in vitro and in vivo.98 Further observations support the inclusion of IL-7 in vaccine-based therapeutic strategies currently being developed for cancer therapy and suggest that the mechanisms of T-cell stimulation by IL-7 may be unique for IL-7 compared with either IL-2 or IL-12 stimulation. It is therefore likely that combinations of these cytokines may lead to enhanced anti-tumour effects. The numerous effects of IL-7 on T cells and its ability to stimulate antigen-specific responses suggest that IL-7 may be useful in the treatment of HIV-infected patients either by direct administration or through the use of IL-7 as an adjuvant in HIV vaccine trials. On the down side, stimulation of I3 lymphopoiesis by IL-7 could result in progression of certain leukaemias. and lymphomas and there is concern that prolonged stimulation by IL-7 could induce new lymphoid tumours. These concerns derive from studies of transgenic mice overexpressing IL-7 which developed both B- and T-cell lymphomas. lo6 Furthermore, IL-7 increases tumour cell proliferation during in vitro culture of human chronic lymphatic leukaemia and acute leukaemia cells. These observations, when taken together, suggest that haematological malignancies may not be appropriate targets for clinical trials of IL-7 and that IL-7 administration in any trial should be for a limited duration.

INTERLEUKIN-12

IL-12 is a pleiotropic cytokine formerly termed cytotoxic lymphocyte maturation factor (CLMF) or natural killer-cell stimulatory factor (NKSF). The cDNA was cloned by two groups in 1991.‘07J08 Unlike most cytokines, IL-12 has a unique heterodimeric structure composed of two chains, encoded by two different genes and both genes are necessary for production of biologically active IL-12. The IL-12 receptor belongs to the haemopoietic receptor superfamily and shows strong homology to gpl 30.1°9This cytokine plays a pivotal role in the induction of a Thl response and, therefore, cellmediated immunity.110 In vitro, IL-12 synergizes with IL-2 in augmenting allogeneic cytotoxic T lymphocyte (CTL) responses, lymphokine activated killer (LAK) activity and IFN-y production from peripheral blood lymphocytes. IL- 12 may also stimulate IFN-y production from T and NK cells directly, increase the lytic activity of NK cells and expand activated NK and

T cells (reviewed in”‘). In addition to immunoregulatory functions, IL-12 has potent activity on haemopoiesis. 11*During in vivo studies in mice, IL- 12 was shown to augment NK function, induce IFN-y secretion, promote extramedullary haemopoiesis and enhance generation of an allogeneic response. The effect of IL- 12 administration in infection models has been examined. One of the most extensively studied models is cutaneous Leishmania major. In this model, IL-12 is able to promote a curative Thl response.‘13 IL-12 has also been shown to be crucial in controlling numerous other parasitic and bacterial infections. The most exciting of IL-125 activities is its antitumour effect. In a variety of model systems, IL-12 has been administered either systemically to tumourbearing animals, by direct injection into tumours, or produced locally by genetically engineered fibroblasts.l14 Models of malignant melanoma, reticulumcell sarcoma and adenocarcinoma have all been investigated and all tumours tested have responded to treatment. At the doses used to obtain anti-tumour efficacy the toxicity has been mild. IL-12 has been more effective in many settings than IL-2. Animals cured of their tumour by IL-12 treatment become immune to rechallenge with the same tumour cells suggesting the potential value of IL-12 as an adjuvant for tumour vaccines (reviewed in’r5). Although most of the anti-tumour activity of IL-12 appears to be mediated through enhanced production of IFN-y by T and NK cells, treatment of tumour bearing mice with IFN-)I alone does not result in the same degree of efficacy, suggesting that further mechanisms may be involved. Clinical trials of IL-12 were initiated in 1994. The most closely studied disease has been HIV/AIDS in which IL-12 production is severely impaired. In this disease, in vitro studies have confirmed that addition of IL-12 to cell cultures restores cellular immune function.l15 The early trials of IL-12 in patients with cancer or AIDS looked both promising and safe. However, during phase II trials, some treated renal cancer patients suffered severe toxic effects leading to at least one death.“‘jJ17 It is known that high doses of IL-12 may synergize with certain viral infections to increase TNF-a production to the detriment of the host. Alternatively, another potential problem is the induction by IL-12 of autoimmune phenomenon in which a Thl response is contributory. The mechanism of toxicity seen in these trials has never been fully elucidated. In summary, IL-12 has considerable potential as an immune modulator for use in vitro and further investigation of this cytokine for the immunotherapy of cancer is certainly warranted.

Cytokines at the research-clinical interface

CONCLUSION The more recently identified cytokines discussed in this review offer a wide range of possible applications for clinical practice in the future. To optimize the mobilization and harvesting of PBPC, it may be necessary to administer early-acting cytokines such as SCF or flt3 ligand in the first days following chemotherapy and to add G or GM-CSF with or without thrombopoietin later. This strategy should limit the toxicity of these cytokines by using each at a relatively low dose and should encourage primitive progenitors to proliferate and hence produce target cells for the later-acting cytokines. IL- 11 offers considerable promise both for its effects on megakaryopoiesis and on the gastrointestinal epithelium. Following high-dose chemotherapy, with or without BMT, the strategy should be to use the early acting cytokines first followed. later by the lineage committed cytokines. Combinations of cytokines including SCF, flt3 ligand, IL-3 and IL-6 have shown potential for the ex vivo expansion of haemopoietic stem cells and for gene transduction protocols. Finally, the immunomodulators IL-4, IL-7, IL-lo; IL-12 and IL-1 3 show promise both for in vivo administration and in vitro manipulation for adoptive immunotherapy. REFERENCES

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