Cytokine-induced radiation protection and sensitization

Cytokine-Induced Radiation Protect ion and Sens i t izat ion Ruth Neta and Paul Okunieff

Cytokines, hormone-like proteins that are produced by stimulated cells and tissues, serve as intercellular mes- sengers. The cloning and large-scale production in a recombinant form of an expanding number of cytokines in the past decade has permitted investigations aimed at assessing the benefit they may provide in preserving and restoring functions of tissues compromised by irradiation. This review focuses primarily on the preclinical and clinical findings of a number of radiation and chemotherapy studies in which cytokines were found to protect, restore, or at times harm the hematopoietic and gastrointestinal tissues, as well as lung and liver, against

cytotoxic therapies. Included are the myelorestorative effects of cytokines, their application in mobilization of peripheral blood progenitor cells for bone marrow transplantation, and their myeloprotective effect when given before irradiation. Studies indicating the importance of the treatment schedule, and in some cases their contrasting effects on different tissues, are included. The insights gained from such studies into the mechanisms of regulation by cytokines of radiation-induced damage are discussed. Copyright �9 1996 by W.B. Saunders Company

C hemical modifiers of radiation response have been studied for several decades, with the goal

of selectively protecting normal tissue from harmful effects while enhancing tumor cytotoxicity. Almost a decade ago it was observed that administration of a proinflammatory cytokine interleukin (IL)-I before irradiation protects a substantial fraction of lethally irradiated mice from death. 1 The concurrent finding that IL-1 inhibits the growth of a number of tumor cell lin~s 2 raised hopes that use of IL-1 and similar cytokines might allow for major increases in dose intensity of radiation therapy while simultaneously protecting the normal tissues. The cloning and large- scale production in a recombinant form of an expanding number of cTtokines has permitted preclinical and clinical investigations aimed at assessing the benefit they may provide in preserving and restoring functions of tissue compromised by irradiation.

Cytokines are hormone-like proteins, produced by stimulated cells and tissues, that serve as intercellular communicators. Several families of cytokines have been identified and cloned in the past two decades, and their characteristics and modes of action are becoming rapidly understood. Many cTto- kines are pleiotropic, ie, they act on multiple cells and tissues and produce a broad range of effects. For example, IL-1, tumor necrosis factor (TNF), and IL-6, all affect hemopoiesis, brain and liver function,

From the Ojfice of International Health Programs, Department of Energy, Germantown, MD, and the Radiation Oncology Branch, National Cancer Institute, Bethesda, MD.

Address reprint requests to Ruth Neta, Ojfice of International Health Programs, Department of Energy, EH-63, 270CC, 19901 Germantown Rd, Germantown, MD 20874-1290.

Copyright �9 1996by W.B. Saunders Company 1053-4296/96/0604-0007505.00/0

in addition to vascular, muscle, and other tissues. 3 Other cytokines, such as interleukin-3 (IL-3), granulocyte colony-stimulating factor (GM-CSF), granulocyte-macrophage colony-stimulating factor (GM- CSF), macrophage colony-stimulating factor (M- CSF), erythropoietin (EPO), thrombopoietin (TPO), or stem cell factor (SCF) affect primarily proliferation and differentiation of hematopoietic cells at various stages of their lineage progression. 4 The immune system is influenced byyet another group of cytokines, including IL-2, IL-4, IL-5, IL-7, IL-12, IL-13, and IL-15 that affect the growth, differentiation, and function of T and B cells. 5 In addition, a number of cytokines have the capacity to down- regulate proliferation of hematopoietic progenitor cells. These include transforming growth factor [3 (TGF[3), macrophage inflammatory protein-la (MIPla), and a family of interferons, IFNot, IFN[3, and IFN3,. 6

Cytokines act via high-affinity membrane receptors, complex in structure, that actively participate through a signal transducing domain, in transmit- ting signals. The signaling by the cytokine-receptor complex leads to stimulation of a diverse range of cell functions including production of cytokines, growth and proliferation, differentiation, motility, death by apoptosis, and growth inhibition. The induction by cytokines of further production of cytokines is re- ferred to as cytokine cascade, and the commonly observed interdependence on one another and synergy of their effects, as cytokine network.

In the past few years the modes of down-stream signaling by a large number of cytokine-receptor complexes are becoming understood. For example, the reasons for the diverse effects of the same cytokine on various cells are gradually becoming

306 Seminars in Radiation Oncology, Vol 6, No 4 (October), 1996.'pp 306-320

Cytokine Radioprotection and Sensitization 307

clear. This diversity depends on the type of signaling receptor, and/or components of the signal transducing pathway engaged in a given cell, thus leading to the subsequent activation of different transcription factors. For example, better understanding of signaling pathways explains the seemingly paradoxical observations that TNF may lead to apoptosis of some cells, yet induce proliferation or differentiation of others. 7

This review will focus primarily on the preclinical and clinical findings of a number of radiation and chemotherapy studies. The cytokines that will be discussed include hematopoietic growth factors, found to protect, restore, or at times harm the hematopoietic and gastrointestinal tissues, as well as lung and liver against cytotoxic therapies. Although emphasis will be given to their role in damage induced by ionizing radiation, because most of the clinical studies were conducted with chemotherapeutic agents, some of these will be reviewed. The insights gained from such studies into the mechanisms of regulation by cytokines of radiation-induced damage will be discussed.

Myeloresgoration With Cytokines Toxicity to the hematopoietic tissue is a common complication of standard cytotoxic therapy, including radiotherapy, that limits intensification of therapy. The particular sensitivity of the hemopoietic system to cytotoxic therapies spurred a considerable interest in development of methods to differentially spare the normal hematopoietic tissue.

One widely applied approach for supportive therapy relies on the hemostimulatory cytokines that are known to act directly on hemopoietic progenitors to accelerate their proliferation. Early on these included granulocyte colony stimulating factor (G- CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), and more recently additional hemostimulatory agents, IL-I, IL-3, IL-3/GM-CSF fusion molecule (PIXY321), IL-6, stem cell factor (SCF), thrombopoietin (TPO), IL-I1 and IL-12 are being tested in phase I and 11 clinical trials. 8 TPO is a recently cloned cytokine 9,'~ directed primarily at counteracting thrombocytopenia. In addition, erythropoietin, a hemostimulatory cytokine licensed for treatment of anemia of renal failure, has been tested as a potential means for counteracting the anemia associated with cancer and its therapy. II To this end the majority of the clinical trials conducted with these cytokines involved their use in conjunction with

intensive cytotoxic chemotherapy. The recombinant human forms of three of these cytokines, Escherichia coli-derived G-CSF (filgrasin or Neupogen), (Am- gen, Inc, Thousand Oaks, CA) yeast-derived GM- CSF (sargramostim, Leukin) (Immunex Corp, Se- attle, WA), and erythropoietin (Epo, Epogen), (Procrit, epoetin alfa), (Ortho Biotech, Raritan, NJ) have shown sufficient efficacy to be approved in oncology practice in the United States.

For example, in 1991 G-CSF became commer- cially available in the United States. It is currently used to decrease the incidence of life-threatening infections in patients with febrile neutropenia associated with leukemias and high-dose chemotherapy. The licensing trial involved 199 patients with small cell lung cancer randomized to receive G-CSF or placebo after chemotherapy that consisted of cyclophosphamide, doxorubicin, and e toposide.12 The primary benefit of G-CSF appeared to derive from its ability to shorten the duration of neutropenia, it also reduced the frequency of febrile neutropenia from 57% in control patients to only 28% in patients receiving G-CSF (P < .001). This led to significantly reduced first-cycle hospitalizations for initiation of antibiotic therapy and shorter hospital stays for patients receiving G-CSF. Since the licensing of the G-CSF, its primary use has been to treat chemotherapy-induced febrile neutropenia rather than to prevent its occurrence.

Myelorestorative trials were also conducted with GM-CSF. For example, in one randomized study of 290 patients with small cell lung cancer undergoing cyclophosphamide-based chemotherapy, GM-CSF therapy decreased the duration of neutropenia and decreased the requirement for antibiotic therapy during cycle 1.13 Both GM-CSF and G-CSF have been used to facilitate chemotherapy dose intensification. For example, GM-CSF '4,15 and G-CSF 16,17 have been shown to accelerate neutrophil recovery and to decrease the duration of infections in patients undergoing autologous bone marrow transplantation (Aaagr).

Only a limited number of trials involve development of cytokines as adjuncts to radiotherapy. One randomized trial tested G-CSF as support for clinical irradiation. 18 Compared with controls, white blood cells (WBCs) were maintained at higher level, and fever duration and antibiotic use were lessened in G-CSF treated groups. However, significant systemic myelosuppression from localized irradiation is rare if less than 40% of the marrow is treated. The advantage of G-CSF may only be seen in patients undergo-

308 Neta and Okunieff

ing salvage radiation therapy after multiple courses of chemotherapy have failed.

Application of Cytokines for Peripheral Blood Progenitor Cell Mobilization

After the first successful peripheral stem cell transplants reported 9 years ago, a new technology for hemopoietic support of patients undergoing high- dose cancer therapy was initiated. Recently, myeloid growth factors have been used to stimulate mobilization of progenitor cells into the circulation. Thus, therapy with G-CSF or GM-CSF, either alone or in combination with chemotherapy, is associated with the increased appearance of the progenitor cells in the peripheral blood. These progenitors can be har- vested by leukophoresis and can be used to rescue the marrow after myeloablative therapyJ 9-29

Compared with historical controls, the use of peripheral blood progenitor cells (PBPC) appears to be superior to autologous bone marrow transplantation (ABMT), with a shorter duration of thrombocytopenia and decreased platelet transfusion requirement, as well as shortened neutropenia, when followed by the cytokine therapy. Current trials explore G-CSF or GM-CSF in combination with SCF or IL-3 without preceding chemotherapy, s~

In a primate model, the effect of a single intravenous injections of rh-GM-CSF, rh-IL-3, rh-IL-l[~, rh-G-CSF, or rh-PIXY 321 on hemopoietic stem cell concentrations in the blood were compared. 3~ When administered as a single dose, only IL-113 significantly increased the concentrations of hemopoietic stem cells. No toxicitywas noted with this procedure. None of the remaining cytokines tested caused a significant increase in the peripheral blood hemopoietic stem cells. Autologous transplantation with a limiting number of IL-1 mobilized peripheral blood mono- nuclear cells conferred a survival advantage to recipi- ents compared with controls.

Myeloprotection With Cytokines

Delivery of the hemopoietic growth factors after cytotoxic therapy is unable to completely ameliorate myelotoxicity and does not prevent a nadir in WBC. As an alternative, transplants with PBPC have a number of drawbacks. One is the potential for preserving and transplanting clonogenic tumor cells. Another is the failure to eliminate the severe bone

marrow aplasia that typically occurs 10 to 14 days after the transplantation. These limitations continue to spur experimentation into the possibility of selectively providing in vivo protection to bone marrow cells betbre administration of the cytotoxic radiotherapy and chemotherapy.

Preclinical Studies

Experiments in the 1950s showed that administration of inflammatory bacterial lipopolyscharide (LPS), prior to lethal irradiation of mice, promoted survival and subsequent recovery of hemopoietic cells, 32 Simi- lar protection from myelotoxicity caused by radiation therapy was achieved by administration of the proinflammatory cytokines IL-1 and TNF. 1,33 Conversely, protection conferred by LPS was blocked by anti-IL-I and anti-TNF antibodiesP 4 These results provided direct evidence that these cytokines serve as endogenous radioprotectors.

Of the many cytokines that have been tested for their survival promoting effect when given as a single injection within an optimal time of 18 to 24 hours before irradiation, only IL-1, TNF, SCF, and IL-12 acted alone to protect mice from radiation doses that are otherwise lethal. The cytokines IL-2, IL-3, IL-4, IL-6, IL-11, GM-CSF, G-CSF, M-CSF, LIF, IFN',/, and TGF13 did not promote survival of lethally irradiated mice. 35 In fact, administration of IL-6, TGFI~, or IFN hours before irradiation can result in increased radiosensitivity in whole-body irradiated mice (Fig 1A, C)P 4,36 This line of experimentation has revealed several insights into the bases of protection of mice from lethal hemopoietic syndrome.

A Radioprotect

wm

IL-1, ~-z4to-~Sh TNFcz, SCF, 11.-12 aFGF, bFGF

B Chemo-sensitize 5-FU

Chemo

IL-1 ~ -z4 to -~8 h

c Radiosensitize D Chemo-protect

WBI ~ Chemo

IL-6~ IFNc~, IFNI~

Figure 1. Cytokine-mediated modification of radiotherapy and chemotherapy. A, radioprotect, B, chemosen- sitize; C, radiosensitize; and C, chemoprotect.


Pree l in ica l Studies With IL-1 and TNF as Protectors Against Radiation Therapy Myelotoxic i ty

IL-I and TNF are two proinflammatory cytokines with biological effects vital to host defenses on the one hand, but contributing to morbidity on the other hand. 3 Thus, their administration to patients leads to desirable as well as toxic effects. The rapidly developing understanding of their structure-function relationship, and of the downstream events of their action in different cells and tissue, aids in identifying their role in each of these processes. Such knowledge in turn should lead to developing means of intervention to harness the beneficial effects while diminishing the undesirable effects of these cytokines.

In most murine strains that were tested, the dose modification factor (DMF) for IL-1 was greater than for TNF. Single doses of 100 to 300 ng of IL-1 given 20 hours prior to radiation had a greater protective action than 5 ~,g of TNF (Figs 2A and 3A). The combined action of these cytokines resulted in synergistic effect (Fig 3C), indicating that the two molecules employ radioprotective pathways that are in part distinct. 33 The combined effect of IL-1 and TNF was also more protective than the optimal dose of bacterial LPS. Conversely, blocking the activity of IL-I and TNF with the specific antibody, in LPS- treated mice, revealed a radiosensitizing effect of LPS. 33 Although there are several possible explana- tions for radiosensitization, perhaps LPS-induced cytokines such as TGF[3, IL-6, IFNot, and IFN~ contributed to the effect.

A Radioprotect ive

wel IL-1 ~ -z4 to -iS h

DMF 1,2

WBI

IL ' I ~ -24 to -18 h DMF 1.3 to 1 .S

& TNF~, SCF

c Radiosensit izing WBI~

before or after

antl-lL-1 antl-lL-6 anti-TNFa anti-SCF

B Non-Radioprotect ive

we, ~, IL-1 ~ ~-72 and -24 h

WBI

IL-1 ~ -48 h

WBI

IL-1 ~ -z4 to .18 h & IntI-TNF,

antl-lL-1, anti-c-Kit, =ntl-lL-6

F i g u r e 2. Interleukin-I (IL-1) mediated WBI radioprotection. A, radioprotective; B, nonradioprotective; and C, radiosensitizing.

A R a d i o p r o t e c t Marrow

WBI

TNF~ ~-Z4to- leh

B Radiosensitize Bowel

WBI

TNF~ ~ -z4 to -18 h

Increased C Radioprotection

WBI ~,

TNF~ + IL-1

Figure 3. TNF mediated WBI radiomodification. A, radioprotect marrow; B, radiosensitize bowel; and C, increased radioprotection.

Complex interactions of cytokines occur in vivo. For example, despite IL-6 sensitizing mice to radiation lethality when given alone, coadministration of this cytokine with suboptimal doses of II.,-I resulted in synergistic radioprotection. 36 Conversely, blocking IL-6 abolished IL-I- and TNF-induced radioprotee- tion. 37 In addition, anti-TNF antibody abolished IL- I- induced protection and anti-IL-1 antibody similarly blocked TNF-induced radioprotection (Fig 2B).34 Be- cause IL-l and TNF induce one another and both induce II.,-6 production, it is apparent that the interaction of IL-1, IL-6, and TNF in vivo is required for optimal radioprotection. Even in mice not treated with cytokines, injection of antibody to IL-I, IL-6, or TNF resulted in LDs0/30 doses of radiation becoming lethal to 100% of mice, indicating that the endogenous production of each of these cytokines contrib- utes to normal radiation tolerance (Fig 2C). 34,37 These examples amply illustrate the need for an understanding of the cytokine cascades and provide evidence for their function in a network.

Both IL-I and TNF stimulate production of a cascade of hemopoietic growth factors, including G-CSF, GM-CSF, IL-6, and IL-3, as well as platelet- derived growth factor (PDGF).3,38-40 It is possible that the induction of these myeloproliferative growth factors accounts for the myelorestorative action of high doses of IL-I and TNF given after radiotherapy. 41 In addition, it was shown that G-CSF and GM-CSF given before lethal irradiation of mice can synergistically cooperate with suboptimal doses of IL-1 to enhance survival. 33 Evidence discussed previously shows that in the cascade of cytokines induced by IL-1 and TNF are also cytokines detrimental to radioprotection. Their neutralization may further enhance the radioprotective effects ofIL-I and TNF.


A very important consideration for using TNF~ in cancer therapy stems from the observation that it is induced in tumor cells by ionizing radiation and together with radiation enhances synergistically death of these cells. 42,4a This interaction was shown for human soft tissue and bone sarcomas, squamous cell carcinomas, and myeloid leukemia. In contrast, normal human fibroblasts showed no evidence of similar sensitization to radiation by TNF. Because hydroxyl radicals are required for maximal cytotoxicity of radiation and TNF, differential production of scavenging molecules may account for different cell responses. Indeed, reduction of the cell killing in anaerobic conditions, or by induction of mitochondrial enzyme manganese superoxide dismutase (Mn- SOD) and free-radical scavengers, provided support for this hypothesis. 44-46 Conversely, hydroxyl radicals were not produced in cell lines resistant to TNF killing. Furthermore, inability to produce MnSOD rendered cells more susceptible to TNF killing and radiation. Conversely, transfection of MnSOD gene to cells conferred increased resistance to ionizing radiation. 47

The tumor sensitizing effects of TNF together with its ability to protect animals from radiation lethality make it a desirable candidate for gene radiotherapy. Radioresistant tumors were injected with liposomes containing a chimeric genetic con- struct that included radiation inducible Egr-1 pro- moter sequences linked to TNF cDNA. 48 Radiation of such genetically altered tumors in mice resulted in TNF upregulation in tumors that paralleled a significantly reduced mean tumor volume.

The Importance o f Treatment Schedu le

Interleukin-1 protects the hemopoietic system of mice against a wide range of cytoablative therapies including ionizing radiation, 1 as well as drugs such as cyclophosphamide, 5-fluorouracil (5-FU), mafos- famide, or doxorubicin. 49-51 However, to achieve such protection, the schedule ofIL-1 administration needs to vary, ie, administration of a single dose of I L l within 18 to 24 hours before irradiation is necessary for radioprotection, 1 whereas administration of multiple daily doses ofIL- 1 (7 days) is required to protect against chemotherapeutic drugs (Fig 1A and D). 49-51

I L l is a potent in vivo stimulator of hemopoiesis. 3,8 Specifically, IL-1 synergizes with hemopoietic growth factors to promote the proliferation of stem and progenitor cells. 52,53 The effects of I L l are

amplified by its capacity to induce hematopoietic growth factors ~9,4~ and to up-regulate their receptors on bone marrow progenitors. 54 Several hours after administration of a single dose ofIL-1, the number of hemopoietic progenitors in the bone marrow increases and reaches a maximum after 48 hours. 55,56 These early findings suggested that an expansion of progenitor cells may be the basis for the myeloprotective action of IL-1. However, mice receiving irradiation 48 hours after IL-I treatment are not protected from death (Fig 2B), 57 indicating that the mere increase in the numbers of progenitor cells is not sufficient for IL- 1 radioprotection.

The kinetics of the radioprotective effect of I L l suggest that the mechanism of radioprotection in- volves the cycling of progenitor cells and the increased radioresistance of the cells in the late S- phase of the cell cycle. 58"6~ Indeed, administration of IL-1 18 hours before irradiation results in optimal radioprotection and coincides with increased sensitivity of progenitor cells of various lineages to hydroxy- urea (HU). 61,62 Because HU is selectively toxic for cells in the S phase, these results imply that I L l induced progenitor cells to progress to the S phase.

In such cases, however, protection by IL-1 of bone marrow cells against chemotherapeutic drugs that are cytoablative to cycling cells would be paradoxical. Using 5-FU, which spares early progenitor cells because of their quiescence but is highly toxic to cycling cells, 63-65 it was shown that pretreatment with a single dose of IL-1 resulted in death within 10 to 14 days of 5-FU-treated mice. 66 The death occurred only when IL-1 was given 18 hours, but not at 4 or 48 hours, before administration of sublethal doses of 5-FU. The death was caused by hemopoietic failure as evidenced by only 2% of primitive hematopoietic progenitor cells surviving in IL-1/5-FU as compared with 5-FU-only treated mice (Fig 1B). Apparently, IL-1 induced the remaining 98% of these normally 5-FU resistant, slow proliferating, or resting cells to cycle, perhaps synchronously reaching S phase at 18 to 24 hours. On the other hand, at an interval of 48 hours, IL-1 no longer sensitized mice to sublethal doses of 5-FU and did not affect significantly the numbers of surviving progenitor cells, suggesting that the IL-1-induced cycling effect was transient.

Importantly, in contrast to radioprotection by pretreatment with a single dose of I L l at 18 to 24 hours, two injections of IL-1 48 hours apart, at 72 and 24 hours before irradiation, abrogated radioprotection (Fig 2B). Likewise, the killing of progenitor cells by 5-FU was greatly reduced when two injections of

Cytokine Radioprotection and Sensitization 3 1 1

IL-1 48 hrs apart were administered before 5-FU. These findings suggest that the 48 hours pretreatment with IL-1 results in abrogation of the ability of the early progenitors to cycle in response to subsequent IL-I challenge. This effect may be based on presence of molecules inhibitory to hemopoiesis. Indeed, within 36 to 48 hours after IL-I treatment TGF[3 mRNA and protein were increased. 66

It was previously observed that TGF[3 sensitized mice to radiation lethality. 33 TGF[3 has been reported to be a potent inhibitor of the cell cycle for many cell types, 67,68 acting through the reduction of the expression of multiple growth factor receptors, including c-kit, 69-74 a receptor for SCF expressed on early hematopoietic progenitors, SCF production, 75 and through activation of cell cycle inhibitors. 76 TGF[3 inhibited the in vitro growth of primitive progenitor cells (HPP-CFC), whereas the growth of more differentiated granulocyte progenitors (G- CFU) was stimulated. 77 Interestingly, five daily injections of IL-l resulted in highly granulocytic bone marrow (BM) (R. Neta, unpublished results, 1993). Furthermore, upregulation and shedding of decoy- like type II IL-1R that interacts with IL-1 may result in tachyphylaxis, that indeed has been observed in patients receiving multiple injections of IL-1.

In addition to TGF[3, other factors induced by IL- 1 and inhibitory to cycling and proliferation ofhemato- poietic progenitors may be involved. For example, prostaglandin and TNF, both induced by I L l , were reported to inhibit the growth of progenitor cells. 7a'8~ Repeated injections of IL-1 lead to the appearance of a serum factor that inhibited colony formation, and was partially neutralized by antibody to TNF. TM More recently, IL-1 was shown to first up-regulate and then down-regulate GM-CSF production in human fibroblasts. The down-regulation was associated with the production of prostaglandins. 81 Thus, a cascade of cytokines and other mediators induced by I L l in- cludes positive as well as negative regulators of cycling of primitive progenitors.

Together, these findings suggest that the myeloprotective effects of IL-1 against ionizing radiation and cytoablative chemotherapeutic drugs are mediated by distinct mechanisms. The mechanisms of cytokine action are complex and include induction and/or inhibition of progenitor cell cycling. Sensitiza- tion and protection against radiation and cytotoxic drugs are highly schedule- and dose-dependent and probably result from temporal cascades of cytokines that are induced by the different treatment sched- ules.

Clinical Experience With IL-1

The diverse effects of I L l are relevant to the experience with this cytokine in phase I and II clinical trials. In all of these studies IL-1 was administered by intravenous infusion once daily for a number of consecutive days. For example, recombinant IL-la and IL-I[3 were given to patients over a 15-minute period daily for 7 consecutive days. s2 Patients with a variety of solid tumors, including metastatic melanoma, sarcomas, advanced carcinoma of the lung, breast, ovary, colon, and head and neck were evalu- ated. The maximum tolerated dose (MTD) oflL-1 in cancer patients was determined to be a relatively low dose of 0.3 Ixg/kg of either IL-la or ILl[3. Patients experienced flu-like symptoms including chills, fever, some nausea and vomiting, mild fatigue, headache, myalagias, arthalagias, and somnolence. None of the patients experienced a severe capillary leak syndrome commonly observed with IL-2 or TNFa therapy. Dose limiting toxicity of IL-1 consisted primarily of the development of hypotension beginning by I to 3 hours after and lasting up to 50 hours after treatment. Another effect of I L l therapy consisted of lowering the serum cholesterol and increasing the serum triglycerides, probably based on the inhibition of lipoprotein lipase. Tachyphylactic diminution of responses occurred after repeated administration of I L l . However, even at the relatively low doses ofIL- 1 tolerated by man, the patients showed significant increases in peripheral blood granulocyte counts and bone marrow cellularity.

IL-I increased neutrophil counts 4 hours after treatment, consistent with the demargination effect. The increase of bands in the peripheral blood indicated that IL-I also caused an egress of cells from the maturation-storage compartment of the marrow. The patients also showed increased numbers of bone marrow megakaryocytes and a 70% increase in their platelets count 1 to 2 weeks after IL-I therapy.S2 This observation led to an evaluation of the capacity of IL-1 to ameliorate carboplatin-induced thrombocytopenia, s3 IL-1 was found to accelerate platelet recovery and shorten the duration of thrombocytopenia when it was given for 5 days starting 24 hours after high-dose carboplatin treatment.

In patients with normal hemopoietic function, IL-113 administration was associated with significant neutrophilia and a striking increase in platelet count. 84 In contrast, in four patients with refractory aplastic anemia, treatment with IL-l[3 did not stimulate hemopoiesis, s5 A recent multicenter phase I study


used IL- 1 [3 in patients with bone marrow failure. 86 In this study IL-I[3 was administered as a 30-minute intravenous infusion once daily for up to 5 consecutive days. Nineteen patients with severe bone marrow failure received 60 courses of IL-I[3 in doses ranging from 0.02 to 0.5 bg/kg. Five of these patients had autologous bone marrow transplants, seven had allogeneic BMT, six had idiophatic aplastic anemia, and one had chronic myeloid leukemia. Toxicities included fever (89%), chills (85%), hypertension (89%), hypotension (57%), and headache (95%). No complications were life threatening, and all either spontaneously resolved or were managed pharmaco- logically. In eight of the patients there was an acute, transient increase in neutrophil counts, but only two patients had a transient increase in platelet counts.

Together, the above studies show IL-1 has dose- dependent toxic side effects. In general, most studies in patients have not shown the striking hemopoietic effects that were observed in murine models. There are several potential reasons for the reduced response. Based on the results with animal models, none of the clinical studies delivered I L l at the required schedule. For example, multiple daily injections rather than a single injection were not radioprotective in animals. Furthermore, chemoprotection was highly dependent on the specific drug and IL-1 delivery schedule. Other differences between mouse and human beings include possible differences in cytokine receptor expression, and the balance between proliferative and antiproliferative cytokines induced by IL-1. Resolution of these questions are needed to optimize the clinical utility ofIL-1.

Myeloprotec t ion With Stem Cell Factor: Precl in ical Studies

Stem cell factor, also known as Kit ligand and its recepto r c-Kit, are important for normal development of hematopoietic cells. Strains of mice with mutations in the steel (SI) and white spotting (W) loci coding for SCF and c-Kit respectively show hematopoietic abnormalities and increased sensitivity to ionizing radiation. 87,88 SCF treatment before radiation protects normal mice from radiation lethal- i ty . 89 Conversely, antibody to its receptor, c-Kit, given to LDs0/30 irradiated mice, leads to 100% lethality. Likewise, treatment with this antibody blocked en- tirely LPS and IL-1-induced radioprotection. 9~ These experiments combined with those described in previ- ous sections suggest that like IL-1, IL-6, and TNF, normal function of SCF and its receptor are required

to achieve maximal protection of bone marrow from ionizing radiation.

Of particular interest was the observation that the coadministration of IL-1 and SCF to mice, in a single dose at 18 to 24 hours before lethal irradiation, was synergistic in protecting mice from death} 7 This protection was associated with a substantial increase in the numbers (fortyfold) of hemopoietic progenitor cells recovered within 1 and 4 days after irradiation, indicating that these cells probably survived the radiation insult. Anti-SCF antibody blocked IL-1- induced radioprotection, and anti-IL-1 antibody greatly reduced SCF radioprotection, again indicating that endogenous production and interaction in a network of these cytokines is required for optimal protection. SCF, unlike IL-I, does not induce hemopoietic growth factors, such as CSFs or IL-6, nor can it induce the radical scavenging mitochondrial enzyme, MnSOD, 57 thus indicating that its radioprotective effect depends on mechanisms other than radical scavenging. However, like IL-1, SCF injection results in enhanced cycling of hemopoietic progenitors, 91 suggesting that such cycling may be the basis to the radioprotective effect of SCF. In addition, SCF was shown to prevent radiation-induced apoptosis of c-kit expressing mast cell lines. 92 This affect was independent of the phase of the cell cycle, and differed from that of IL-3. Whereas IL-3 was shown to prevent apoptosis by induction of bcl-2 gene expression, SCF did not upregulate the bcl-2 gene. Thus, the radioprotective effect of SCF may be based on this cytokine's ability to stimulate cycling of hemopoietic progenitors as well as prevent their apoptosis.

Myelorestorative Potential of the Interleukin-6 Family of Cytokines

IL-6, IL-11, leukemia inhibitory factor (LIF), on- costatin M (OSM), and ciliary inhibitory factor (CNTF), are multifunctional cytokines that share a common receptor, a gpl30 signal transducing protein. 93 IL-6 and IL- 11 synergize with other hematopoietic growth factors, SCF and IL-3, to promote colony growth and differentiation of hematopoietic lineages. 94 In vitro studies indicated that the increase in platelets by IL-6 and IL-11 may depend on the increase in ploidy of megakaryocytes. 95,96 Administra- tion of either of these two cytokines to nonhuman primates stimulates dose-dependent thrombocyto- sis. 97-99 As discussed above, although IL-6 synergized


with IL-I to protect mice from radiation lethality, given alone IL-6 sensitized mice to radiation.

Both IL-6 and I L l 1 administered to mice after midlethal irradiation promoted survival and restored hematopoiesis, including platelet recovery. 1~176176 Clearly both cytokines can function to accelerate proliferation of bone marrow progenitors and aid in the bone marrow recovery from cytotoxic stress.

The Effects of Cytokines on Gastro intes t ina l Tissue

Experimental evidence in animal models indicates that IL-11, IL-1, and SCF protect the gut from radiation damage (Table 1). IL-11 increased survival and led to a rapid recovery of intestinal mucosa of mice receiving radiochemotherapy (5-FU and irradiation). 1~ I L l 1 was administered daily beginning on the same day as irradiation and resulted in improved survival. The recovery was associated with an increase in the mitotic index of crypt cells. Similarly, daily treatment with II_~l I of mice receiving radiation only resulted in increased survival of intestinal clonogenic crypt cellsJ ~ The Do for these cells in mice given IL-I 1 for 2 days before radiation was 2.0 Gy compared with 1.8 Gy in control mice. The Do was substantially increased when the treatment was con- tinued for 3 additional days after irradiation to 2.3 Gy. The level of protection afforded by posttreat-

Table 1. Radiomodifying Effects of Cytokines

Cytokines Effect of Treatment References

IL- 1 Radioprotect when 1 TNF-a given before 33 SCF radiation (bone 57, 89 IL- 12 marrow) 110 bFGF 118

IL- 1, TNF-a, IFN-~/ Myelorestoration 18 IL-3, GM-CSF, (given after 35

G-CSF < LD95/s0) 35 IL-4, LIF, bFGF 100, 101 IL-6, IL-I 1

IL-6 Radiosensitize 36 TGF-[3 when given 34 IFN-a/[3 before radiation 35

(bone marrow) IL-1, IL-I 1, SCF Protection of the 105, 106

gut 103, 104 107

IL-12, IFN-,/, Sensitization of the 110 TNF~ gut

ment alone was minimal. Of particular interest was the observation that the extrapolation number (N), which is the measure of the shoulder size of the survival curve, was significantly reduced in the post- treated versus pretreated groups. Such a reduction is thought to represent a reduction in DNA repair capacity or an increase in radiation-induced apoptosis.

I L l given to mice 20 hours before total body irradiation modestly protected duodenal crypt cells. However, given 4 to 8 hours before irradiation, IL-1 sensitized crypt cells to radiation. 1~ IL-1 exposure did not substantially alter the slope of the survival curve, but affected the shoulder, with the survival curve offset to the right by 1 Gy at 20 hours before and to the left by 1.28 Gy at 4 hours before irradiation. The protective effect therefore may be based on the repair of sublethal injury. The radioprotective effect of I L l was also reported by Wu and Miya- moto, who did not observe the sensitizing effectsJ ~ Thus, once again, depending on the time of treatment, IL-I may have opposing effects on tissue damage by radiation.

SCF treatment was also shown to enhance the survival ofmouse duodenal crypt stem cells. The dose modification factor for LDs0/6 was 1.28 and was increased from 14.9 Gy to 19.0 GyJ ~ The schedule of treatment required to achieve protection was from 24 to 2 hours before treatment. Although the exact mechanism for this protection remains to be estab- lished, the previously discussed antiapoptotic effects of SCF may be the basis of this protectionY 2

C o n t r a s t i n g Ef fec t s o f IL-12 on H e m a t o p o i e t i c and G a s t r o i n t e s t i n a l T i s s u e s

IL-12 has potent antitumor and antimetastatic activity in a number of murine tumor models, l~ This property, along with the observation that it is a potent stimulator of the early hematopoietic progenitor cells, 1~ suggested that in addition to playing an important role in cancer therapy, it may serve as a myeloprotector. Indeed, administration of II.,-12 within 24 hours before otherwise lethal irradiation protected a significant fraction of mice from lethal hematopoietic syndrome.l l~ The radioprotection was associated with a significant increase in the numbers of hemopoietic progenitor cells found in marrow 3 days after 1200 cGy. IL-12 however, radiosensitized not radioprotected the gastrointestinal tract, as evi-

3 14 Neta and Okunieff

denced in mice that died within 4 to 6 days after receiving IL-12 and 1200 cGy. The gastrointestinal syndrome was documented by gross necroscopy and histological evaluation. Induction of a similar syndrome in mice not treated with IL-12 required doses greater than 1600 cGy. Thus, at doses of radiation at which IL-12 still protects bone marrow cells, it sensitizes the intestinal tract to radiation damage. Whereas protection of hematopoietic cells was abrogated with anti-IL-1R and anti-SCF antibody, the sensitization of the intestinal tract was prevented with anti-IFN~/ and anti-TNF antibody (Fig 3B). Thus, different cytokines are involved in IL- 12 protection of the bone marrow, versus its sensitizing effect on the gut.

Sensitization of the gut epithelial cells by IL-12- induced IFN'y may be associated with this cytokine's ability to upregulate Fas antigen. This has been shown for lymphocytes and for CD34 + hematopoietic progenitor cells. HI Whereas freshly isolated CD34 + cells do not express Fas antigen, stimulation of these cells with IFN~/or TNF~ markedly increased this antigen expression. Ligation of Fas in the presence of IFN"y or TNFo~ induces apoptosis of hematopoietic progenitor cells. It is therefore possible that IL- 12-induced IFN'y and TNF upregulated gut epithelial cell apoptosis via similar mechanisms leading to exacerbated gut destruction in irradiated mice. Con- sistent with radiosensitization effects IFN',/has on the gut epithelial cells, IFN',/ has been used as sensitizer of solid tumors.H2

Radioprotect ion With Acidic and Basic Fibroblast Growth Factors

Basic fibroblast growth factor (bFGF), also known as FGF2, is a member of a family of growth factors with pleiotropic effects including induction of endothelial proliferation and angiogenesis. 113 Several studies reported radioprotection by bFGF of lung endothelial cells and prevention of radiation-induced apoptosis. I14'115 Subsequent reports failed to confirm lung radioprotection, ll6 The differences in experimental results between these studies may be caused by the use of esophageal shielding in the study showing pulmonary radioprotection, or to strain differences of experimental mice. Recently bFGF was also found to radioprotect bone marrow in whole body irradiated C3H mice. 117 The dose-modifying factor was modest at 1.10 to 1.20, as was the dose-modifying factor for lung tolerance in earlier studies. Ha The radiation protection of marrow, based on histological

examination, was attributable to improved recovery of hematopoietic cellularity.l 17

The mechanisms of the marrow recovery remain speculative. CFU-C assay of bone marrow from animals treated with bFGF indicated augmentation of the otherwise flat shoulder of the radiation dose response curve. 118 Similar changes in the radiation dose response curve have also been observed with several other human recombinant cytokines, I~ in endothelial cells in vitro, 12~ and in adrenal carcinoma cells transfected with FGF4.121 One study suggests that this manifestation of potentially lethal damage repair is attributable, at least in part, to prevention of radiation induced apoptosis. 114 In addition to apparent alteration of bone marrow progenitor cell intrin- sic sensitivity, FGF is a costimulator of myeloprolifera- tion122,12s and may radioprotect by altering the growth fraction or the cell cycle distribution of the marrow. The relative importance of various mechanisms of protection in vivo is unknown.

Interestingly, the effect of bFGF differs in different strains of mice. In C3H mice bFGF accelerated the recovery of all hematopoietic lineages. Balb/C mice had more rapid recovery of only the erythropoi- etic and megakaryocyte lineages, and C57 mice showed little if any myeloprotection by preirradiation or postirradiation FGF treatment. These differences in response of various strains may be based on (1) variable pharmacokinetics delivery of FGFs to bone marrow; (2) variation in expression of FGF receptors on hematopoietic progenitor cells; (3) differences in endogenous FGF levels that may be high and satu- rate receptors in some strains; (4) different components of the extracellular matrix may alter release and receptor binding of FGFs and proteoglycans. 124 FGF has been shown to upregulate production of certain cytokines, ie, IL-4, IL-6, IFN 7, but not IL-1, TNF, or GM-CSF and G-CSF. 125-127 Similar to the case of other cytokines, in vivo radioprotection by FGF may be attributable to a complex interplay between various cytokine networks.

Tumor Growth and Fibroblast Growth Factor

The role that FGF plays in angiogenesis may have an important clinical application. In particular, some tumors can produce FGF in large quantities, and FGF measurements in the urine can be used as tumor markers for some cancers, including breast cancer. 128 Tumor aggressiveness is associated with FGF production. Associations between tumor invasiv-


ity, metastatic frequency, and FGF production have been shown for breast cancer, melanoma, Kaposi's sarcoma, and cervical cancers. 129"13t In addition, experimental human MCF-7 breast tumors, normally requiring hormone supplementation in female mice to grow, when transfected with FGF4 or FGFI (acidic FGF) become hormone independent. Such transfected tumors can grow in male mice and spontaneously metastasize from the breast implantation site to the lung and other organs/3~ Also, FGF, when given systemically at high dose, and on a frequent schedule, can augment tumor growth in animal models. 133 Interestingly, lower FGF doses, sufficient to prevent death and maximally radioprotect after whole-body radiation, do not alter tumor growth rate in mice. Conversely, one murine squamous carcinoma tumor line had a mildly improved radiation response after intratumoral FGF injection. The mechanism here may have been a reduction in the hypoxic cell fraction. 1a4,135

FGF therefore plays an important role in angiogenesis, and although not oncogenic itself, is an important contributor to tumor aggressiveness and the malignant phenotype. The discovery that heparins 136 and certain drugs lsT,13a can bind FGF and reduce angiogenesis and tumor aggressiveness has fueled a large effort to develop antiangiogenic drugs to treat cancers.

Many cancer therapies are themselves antiangiogenic. Radiation in patients produces a long-lasting antiangiogenic response that can lead to late damage. To date there are no clinical studies delivering FGF to patients with cancer to prevent antiangiogenic consequences of therapy or to improve wound healing, although it has been recommended) 39 Be- side the concern that FGF might augment tumor growth rate and metastatic frequency, the main reason it has not been more widely studied in human beings is the limited availability of the human recombinant protein and its high price. In animals, where doses over l mg/kg have been given, FGF appears to be nontoxic.

Radiation-Induced Fibrosis and Transforming Growth Factor

Radiation-induced fibrosis is perhaps the most univer- sal late effect of radiotherapy. Clinically fibrosis occurs most commonly in sites that have pre-existing fibroplastic proliferation, such as sites of irradiation, trauma, or surgery. Fibrosis can lead to organ disfunc- tion, strictures, and ischemic damage. Whether pa-

renchymal and vascular damage caused by radiation lead to organ dysfunction and ischemic necrosis followed by inflammation, or whether fibroblast and endothelial proliferation results in dysfunction and necrosis, is unknown.

TGFI3 stimulates fibroblasts and endothelial cells to migrate to sites of injury, where they proliferate and play an important part in wound healing? 4~ TGF[3 stimulates fibroblasts to produce extracellular matrix, including the synthesis of collagen I and collagen m as well as fibronectin. TGF[3 is implicated in the pathogenesis of chronic hepatitis, idiopathic pulmonary fibrosis, systemic sclerosis, mesangial proliferative glomerulonephritis, and cirrhosis after exposure to carbon tetrachloride, i4~ More recently, TGF[3 has been associated with pneumonitis and veno-occlusive disease after chemotherapy-only autologous transplants of breast cancer patients. 143,144 TGF[3 is synthesized and converted from latent to active form rapidly after irradiation of tissues in vivo, and chronically high levels have been associated with radiation pneumonitis in lung cancer patients) 45

TGF[3 is associated with late toxicity to the lung in human beings undergoing bone marrow transplant 14s,~44 and in mice receMng thoracic irradiation. 144,146,147 In certain murine models, animals who respond to radiation have lungs that immunohisto- chemically stain for TGF[3, while those without fibrosis do not) 4s It is not known whether any of the findings are caused by abnormal production of this cytokine after irradiation. If this growth factor is at the root of certain late radiation effects, antibodies or other biological or chemical modifiers of TGF[3 effects could prevent fibrosis.

Conclusions

The impact of cytokines on cells, organs, and whole animals is striking. Serving as intercellular messen- gers, cytokines represent cellular responses to system perturbation, and usually act in a paracrine fashion, at picomolar concentrations. Their induction in a cascade and their network interactions have probably evolved to amplify their beneficial and to minimize their harmful effects. Thus, the current attempts of clinical application of cytokines in pharmacological quantities may be excessive. Cytokines can have synergistic or antagonistic effects, which may at times depend on the cell type and the physiological context of the cell. It may be advantageous to apply their combination at low concentrations, or for short intervals, thus reducing some of their harmful ef-

3 1 6 Neta and Okunieff

fects. W e have p re sen ted evidence to indicate tha t

the i r effects can be dose-, schedule-of-exposure- , and

organ-specific. D e g r e e of effect can also vary wi th

an imal species, and even strain wi thin species. Al-

though there is g rea t expec ta t ion of clinical benefits

based on f inding tha t several cytokines protec t or-

gans and tissues f rom d a m a g e by ionizing radiat ion,

more work at the molecular , cellular, and whole-

an imal level is needed before cytokines can be used to

m a x i m a l advantage .

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Cytokine-induced radiation protection and sensitization