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Protection Against Mucosal Injury By Growth Factors and Cytokines

Protection Against Mucosal Injury By Growth Factors and Cytokines Abstract This article provides an overview of published studies in which growth factors and cytokines were used to modify the sensitivity of intestinal stem cells to a dose of radiation. In these experiments, growth factors were used to manipulate the sensitivity of stem cells in the gastrointestinal tract to reduce the severity of gastrointestinal mucositis in cancer therapy patients. Transforming growth factor β3, interleukin 11, and keratinocyte growth factor were used. All three agents, given according to appropriate protocols, can result in a threefold to fourfold increase in the number of intestinal stem cells that survive a dose of radiation therapy. This result was assessed by using the crypt microcolony assay of stem cell functional capacity. The changes in stem cell survival that were observed resulted in increased animal survival. This increased survival was taken as a surrogate for improvement in patient well-being. The severity of diarrhea, a marker of functional impairment, was concomitantly reduced. Cancer therapy involves a fine balance between the use of a large enough dose of a drug to kill tumor cells and the prevention of damage to the normal tissues of the body, notably the oral mucosa, the gastrointestinal tract, and the hematopoietic system. Damage caused during cancer therapy to the epithelial layer lining the mouth causes the lining to become depleted and ulceration of the mouth to occur (oral mucositis). This makes it difficult for the patient to eat, swallow, or speak and causes pain and susceptibility to infection. A combination of patient discomfort and possible infection can lead to treatment delay or dose reduction, which can result in a less favorable outcome for the patient. Another major dose-limiting tissue is the gastrointestinal tract, whose rapid cell cycle makes it more susceptible to the effects of cytotoxic exposure and to a rapid expression of damage. This damage can be seen in the small intestine within a few days and within a slightly longer period of time in the large bowel. This article will concentrate on the specific problems associated with damage to the normal small intestine. The small intestine is a constantly renewing tissue, continuously replacing cells that are lost in the lumen of the intestine. This renewal is achieved by the production of new cells in the crypts by the stem cells and their progeny, arranged in an amplifying transit lineage of six to eight generations. Under steady- state situations, there are thought to be between four and 16 actual stem cells that are located near the base of the crypt. However, if the system is damaged (e.g., by radiation) an acute response occurs, causing some cells in the lower region of the crypt, possibly the stem cells, to die via apoptosis. The stem cells, probably along with some early transit generation cells, will recognize the damage and undergo rapid cell division to regenerate the crypt and, hence, the tissue by clonal growth. The number of clonogenic cells per crypt is thought to depend on the level of damage induced, but it could be as many as 30 cells per crypt (1). Some crypts will become reproductively sterile, will be unable to initiate this regeneration response, and will disappear within approximately 48 hours. The time sequence for this response is as follows (Fig. 1). In the first 2–3 days following a dose of radiation (e.g., a 14-Gy dose), some crypts can be seen to be regenerating (forming microcolonies) alongside other crypts that have been reproductively sterilized. On day 4 following radiation therapy, those crypts that have survived the radiation damage will be approximately 1.5 to two times bigger than a normal crypt, and the sterilized crypts will have disappeared. These large, regenerating crypts will then start to split or bud into several crypts (2). Approximately 14 days after irradiation, the regenerating crypts and foci will have grown visible to the naked eye as macrocolonies (3,4). In certain cases, budding will continue until the whole of the intestine has been regenerated and the normal architecture restored. There are advantages in protecting the clonogenic stem cells from damage, since they are the key to the survival of an individual crypt. The number of crypts that survive following cytotoxic damage determines how intact the intestinal mucosa is and, hence, how well an animal or a patient can survive the damage. The number of surviving crypts plays a pivotal role in the competitive race between depopulation and ulceration and regeneration. Alternatively, a stimulatory factor given before cytotoxic exposure could increase the number of stem cells per crypt that are subjected to the cytotoxic insult. A possible method of protecting the clonogenic stem cells might be to manipulate them by using growth factors. This could be achieved by using a growth factor that will take the stem cells out of the cycle, making them more resistant to the cytotoxic damage, and ensuring the survival of more stem cells capable of regenerating any damaged tissue. Finally, giving, after cytotoxic insult, a growth factor that could increase the rate of proliferation or initiate regeneration earlier may help in speeding up the regeneration process. A combination of various protocols could give maximum protection for the epithelium. Various experiments to manipulate clonogenic stem cells to protect against cytotoxic damage have been carried out, but this review will outline work that has used transforming growth factor β3 (TGF-β3), interleukin 11 (IL-11), and keratinocyte growth factor (KGF), using 10–12-week-old (C57BL/6 × DBA/2)F1 (BDF1) mice. All experiments were performed within the regulations of the U.K. Scientific Procedures Act (1986). Transforming Growth Factor β3 TGF-β3 is a known inhibitor of some epithelial cell proliferation (5,6) by preventing cell cycle progression and accumulating cells in G1 or G0. For clonogenic stem cells in the intestine, this might render them more resistant to the cytotoxic damage, leaving more clonogenic stem cells to start the regeneration process. To assess the capacity of TGF-β3 to protect the intestine from radiation damage, we used the crypt microcolony assay as described by Withers and Elkind (7). This is an accepted test for crypt stem cell functional capacity. A standard dose of 2.5 mg TGF-β3 was administered intraperitoneally to male BDF1 mice 24, 8, and 4 hours before and then once immediately after irradiation. Animals were culled and samples of small intestine were taken 4 days after irradiation. The number of surviving crypts per circumference (a unit length) was counted for 10 circumferences per mouse. Data were analysed by using the DRFIT program (8), which allows curves to be fitted and tests the statistical difference between treated and vehicle control groups by using a variance-ratio F test. As shown in Fig. 2, a, four injections of TGF-β3 administered once immediately after and again at 4, 8, and at 24 hours before radiation therapy compared with the vehicle control shows a statistically significant shift to the right of the treated curve (9) (P<.001). This indicates that there are four times more clonogenic stem cells surviving at 15 Gy in the TGF-β3-treated group than in the control group. It is interesting that if an additional dose of TGF-β3 was administered 4 hours after irradiation (–4, –8, –24, 0, and +4 hours), then the level of protection afforded is reduced (data not shown). TGF-β3 administered intraperitoneally after radiation therapy caused crypts to become sensitized, with only approximately one third of the crypts surviving, in comparison with the vehicle control (data not shown). This finding demonstrates the importance of the use of a growth factor in the correct protocol. These studies demonstrated that the small intestinal clonogenic stem cells can be protected from radiation damage. How does this finding relate, however, to the patient's well-being in cancer therapy? To answer this question, we conducted a series of experiments to study animal survival over time after a dose of radiation. Groups of 20 animals were pretreated with TGF-β3 or vehicle, then irradiated with a 15.8-Gy x-ray and observed for 30 days. Animals had their heads, thoraxes, and forelimbs shielded to reduce the complications of oral and hematopoietic damage. Almost 100% of animals pretreated with TGF-β3 survived to 30 days, but only approximately 35% of the vehicle control animals survived (10) (Fig. 3, a). During the 30 days postirradiation, animals were checked twice daily for diarrhea (verified by evidence of wet feces on the fur of the anal region). The duration of diarrhea can be seen on individual animal “lifelines” as shown in Fig. 4. Administration of TGF-β3 before the radiation dose reduces the total number of days during which diarrhea was recorded, suggesting not only an improvement in life expectancy within this experiment but also an improvement in quality of life and a reduction in diarrhea, a surrogate marker of intestinal dysfunction (10). The histology of the intestine of the animals surviving to 30 days is indistinguishable from that of control subjects (Fig. 1). This indicates truly remarkable regenerative capacity, especially when the appearance of the intestine at days 3–5 (Fig. 1) is considered. Interleukin 11 A pleiotrophic cytokine that affects many different systems, recombinant human IL-11 was originally isolated and cloned from an immortalized primate bone marrow stromal cell line (11). Similar experiments to the TGF-β3 study were carried out by using IL-11. A number of protocols were tested with use of the crypt microcolony assay, and these were carried out as follows: protocol A: IL-11 administered subcutaneously at 9 am and at 9 pm, for a total of five injections before irradiation; protocol B: IL-11 administered subcutaneously at 9 am and at 9 pm, for a total of seven injections postirradiation; and protocol C: IL-11 administered subcutaneously at 9 am and at 9 pm, for a total of three injections prior to irradiation and six injections following irradiation. In all cases, IL-11 protected the clonogenic stem cells from radiation damage, but to varying degrees. Protocol A gave good protection with a statistical significance of P<.001, and protocol B was only modestly protective (P = .003), while the best protection was afforded by administering IL-11 subcutaneously both before and after radiation therapy (protocol C; P<.001) (Fig. 2, b) (12), when up to 3.5 times more crypts survived a dose of 16-Gy irradiation in the IL-11 treated groups. Studies examining IL-11 and animal survival over time after radiation therapy did not take into consideration the damage caused to the hematopoietic system. Protocols A and C were carried out with a dose of 12-Gy x-ray irradiation. Animals were observed over the following 30 days. As a result of damage to the hematopoietic and gastrointestinal systems, all of the animals treated with a dose of 12-Gy irradiation died during the first 11 days; however, the IL-11-treated groups survived an average of 2 days longer than the controls (Fig. 3, b) (13). It is unclear by what mechanisms IL-11 protects against cytotoxic damage, but IL-11 given before radiation therapy alters the sensitivity of clonogenic stem cells, making them more resistant; hence, more of them survive. It is uncertain whether IL-11 alters cell cycle progression or has an indirect effect by altering other growth factors. IL-11 protects best when it is given both before and after irradiation, and it may act via different mechanisms before and after irradiation. IL-11 is known to synergize with other factors [e.g., stem cell factor and steel factor (14)] and also may enhance the effects of IL-3 (15). Keratinocyte Growth Factor KGF was identified originally as a factor that stimulated epithelial cells in vitro (16). Further studies demonstrated that it also stimulates both proliferation and differentiation in a number of epithelial cell types when given to healthy animals. This is very noticeable in the gastrointestinal tract (17). It has been observed that administration of KGF has trophic effects on the gastrointestinal tract, increasing crypt depth and villus length (18). KGF also has a particularly pronounced trophic effect on oral mucosa (19). Crypt survival studies with the use of KGF as a protective agent against radiation damage have shown that KGF given for 3 days before irradiation showed a statistically significant protective effect over a range of doses (P<.001) (Fig. 2, c), with three times more crypts surviving a dose of 16-Gy irradiation in the KGF treated group and 3.5 times more crypts surviving after a dose of 14-Gy irradiation than controls (vehicle treated) (18). Use of KGF postirradiation was not found to improve crypt survival. Animal survival studies have also been performed with KGF and 12 Gy of radiation by using postirradiation bone marrow transplantation to protect the hematopoietic system (Fig. 3, c). This experiment showed that 90% of mice pretreated with KGF before receiving 12 Gy of irradiation and a bone marrow transplant survived a 16-day observation period, whereas mice that received only 12 Gy of irradiation and a bone marrow transplant all died during the first 8 days. It seems somewhat puzzling that the best protocol for protection is to give KGF before cytotoxic insult when it is known that KGF is a stimulator. The mechanism by which KGF protects the intestinal system is unknown and could be multifactorial. However, KGF has been shown to have trophic effects on the gastrointestinal tract, increasing crypt size. This may suggest that KGF protects the crypts by proportionally increasing the number of clonogenic stem cells per crypt (18). Indeed, cell proliferation studies have clearly indicated increased stem cell proliferation following KGF treatment, as well as (rather surprisingly) a reduction in transit cell proliferation (20). Stimulatory factors, such as KGF and, possibly, IL-11, may also act as cell survival factors that prevent apoptosis. KGF has also been shown to increase the number of goblet cells in the gastrointestinal tract (17). Goblet cells produce mucins that act as a barrier between the epithelium and the luminal and trefoil proteins that act to defend the gut and can also aid in its repair. One difficulty in making comparisons between the effects of these different agents is that different injection regimens have been used, as have, in some cases, different radiation doses and delivery protocols. However, it is very clear that the sensitivity of the potential clonogenic stem cells in the small intestine of the mouse can be experimentally manipulated by exogenous growth factors or cytokines in an advantageous manner. Such manipulations have been shown to afford overall radioprotection to an animal, and this protection manifests itself in potentially dramatic changes in animal survival and well-being. Conclusions These studies show that statistically significant and, in some cases, dramatic reductions in mucositis can be effected by appropriate manipulation of stem cell sensitivity with the use of growth factors and cytokines. The results presented here are preliminary; extensive additional studies are required to determine the most effective doses and delivery protocols. Many more growth factors and cytokines should be tested, together with combined and sequential use of different factors. With the identification of intestinal-specific regulatory factors, it would seem likely that the sensitivity of the critical stem cells might be even more effectively manipulated. This would reduce the severity of gastrointestinal mucositis even further, improving the quality of life of cancer therapy patients and possibly allowing for dose escalation and improvement in cure rates. Fig. 1. View largeDownload slide Radiation effects on small intestine. (a) (Original magnification ×100 ). The earliest response is an increase in apoptosis 4.5 hours following 8-Gy irradiation. Arrows indicate apoptotic bodies. (b) (Original magnification ×50). A selected area of intestine demonstrating a single large regenerating crypt 4 days after 14-Gy irradiation. Vincristine helps identification by arresting cells in metaphase. Arrowheads = mitosis. Larger arrows indicate two reproductively sterilised crypts that are dying and disappearing. (c) (Original magnification ×40). Day 6 following 14-Gy irradiation. Many areas of intestine are completely devoid of crypts and villi with only a few epithelial cells remaining. (d) (Original magnification ×40.) Section through a normal (control) small intestine showing crypts and villi. (e) (Original magnification ×40). By day 8 following 14-Gy irradiation the few regenerating crypts (see Fig. 1, c) have split (budded) and are starting to form new crypts and villi. (f) (Original magnification ×40.) By day 30 following 14-Gy irradiation the epithelium in the surviving animals is restored to its normal small intestinal architecture, as seen in Fig. 1, d. Fig. 1. View largeDownload slide Radiation effects on small intestine. (a) (Original magnification ×100 ). The earliest response is an increase in apoptosis 4.5 hours following 8-Gy irradiation. Arrows indicate apoptotic bodies. (b) (Original magnification ×50). A selected area of intestine demonstrating a single large regenerating crypt 4 days after 14-Gy irradiation. Vincristine helps identification by arresting cells in metaphase. Arrowheads = mitosis. Larger arrows indicate two reproductively sterilised crypts that are dying and disappearing. (c) (Original magnification ×40). Day 6 following 14-Gy irradiation. Many areas of intestine are completely devoid of crypts and villi with only a few epithelial cells remaining. (d) (Original magnification ×40.) Section through a normal (control) small intestine showing crypts and villi. (e) (Original magnification ×40). By day 8 following 14-Gy irradiation the few regenerating crypts (see Fig. 1, c) have split (budded) and are starting to form new crypts and villi. (f) (Original magnification ×40.) By day 30 following 14-Gy irradiation the epithelium in the surviving animals is restored to its normal small intestinal architecture, as seen in Fig. 1, d. Fig. 2. View largeDownload slide Intestinal crypt survival curves comparing treated groups against respective vehicle controls. All vehicle groups are represented as open circles and all treated groups as closed symbols. (a) Animals were given vehicle or 2.5 mg transforming growth factor β3 (TGF-β3) intraperitoneally at 24, 8, and 4 hours before irradiation and once immediately after irradiation. Values defining the TGF-β3 survival curve are (mean ± SE) D0 = 1.26 ± 0.10, N = 5664 ± 4698 and the values for the vehicle curve are D0 = 1.12 ± 0.83, N = 5294 ± 4492. There is a statistically significant difference between the two curves (P<.001). D0 is the mean lethal dose, the reciprocal of the slope on the exponential position of the curve—it is a measure of the radiosensitivity; N is the back extrapolate to zero dose of the exponential portion of the curve and a measure of the size of the shoulder. (b) Animals were given vehicle or 2.5 mg interleukin 11 (IL-11) subcutaneously 1 day before, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm, for a total of three injections before irradiation and six injections after irradiation. Values for the IL-11 survival curves are (mean ± SE) D0 = 2.30 ± 0.94, N = 110.5 ± 23.7, and the values for the vehicle group are D0 = 1.84 ± 0.67, N = 232.5 ± 52.9. There is a significant difference between the two curves (P<.001). (c) Animals were given vehicle or 12.5 mg keratinocyte growth factor (KGF) subcutaneously once a day for 3 days before irradiation. Values for the KGF curve are D0 = 1.91 ± 0.12, and N = 173 ± 65, and the values for the vehicle control curve are D0 = 1.43 ± 0.06, and N = 886 ± 294. There is a statistically significant difference between the two curves (P<.001). Fig. 2. View largeDownload slide Intestinal crypt survival curves comparing treated groups against respective vehicle controls. All vehicle groups are represented as open circles and all treated groups as closed symbols. (a) Animals were given vehicle or 2.5 mg transforming growth factor β3 (TGF-β3) intraperitoneally at 24, 8, and 4 hours before irradiation and once immediately after irradiation. Values defining the TGF-β3 survival curve are (mean ± SE) D0 = 1.26 ± 0.10, N = 5664 ± 4698 and the values for the vehicle curve are D0 = 1.12 ± 0.83, N = 5294 ± 4492. There is a statistically significant difference between the two curves (P<.001). D0 is the mean lethal dose, the reciprocal of the slope on the exponential position of the curve—it is a measure of the radiosensitivity; N is the back extrapolate to zero dose of the exponential portion of the curve and a measure of the size of the shoulder. (b) Animals were given vehicle or 2.5 mg interleukin 11 (IL-11) subcutaneously 1 day before, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm, for a total of three injections before irradiation and six injections after irradiation. Values for the IL-11 survival curves are (mean ± SE) D0 = 2.30 ± 0.94, N = 110.5 ± 23.7, and the values for the vehicle group are D0 = 1.84 ± 0.67, N = 232.5 ± 52.9. There is a significant difference between the two curves (P<.001). (c) Animals were given vehicle or 12.5 mg keratinocyte growth factor (KGF) subcutaneously once a day for 3 days before irradiation. Values for the KGF curve are D0 = 1.91 ± 0.12, and N = 173 ± 65, and the values for the vehicle control curve are D0 = 1.43 ± 0.06, and N = 886 ± 294. There is a statistically significant difference between the two curves (P<.001). Fig. 3. View largeDownload slide Survival time of animals exposed to 12-Gy (experiment with interleukin-11 [IL-11]) or 15.8-Gy ( experiment with transforming growth factor β3[TGF-β3]) x-rays delivered whole body (IL-11) or abdomen only (TGF-β3). For the keratinocyte growth factor (KGF) experiment, 12-Gy Cs137 was delivered whole body, followed by a bone marrow transplant. All vehicle groups are represented as open symbols, and all treated groups are represented as closed symbols. A) Animals were given vehicle or 2.5 mg TGF-β3 24, 8, and 4 hours before irradiation and once immediately after irradiation. B) Animals were given vehicle or 2.5 mg IL-11 subcutaneously 1 day before irradiation, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm. C) Animals were given vehicle or 5 mg/kg KGF per day subcutaneously on days 2, 1, and 0; that is, before and at the time of (day 0) radiation. PB = partial body, WB = whole body, BMT = bone marrow transplant. Fig. 3. View largeDownload slide Survival time of animals exposed to 12-Gy (experiment with interleukin-11 [IL-11]) or 15.8-Gy ( experiment with transforming growth factor β3[TGF-β3]) x-rays delivered whole body (IL-11) or abdomen only (TGF-β3). For the keratinocyte growth factor (KGF) experiment, 12-Gy Cs137 was delivered whole body, followed by a bone marrow transplant. All vehicle groups are represented as open symbols, and all treated groups are represented as closed symbols. A) Animals were given vehicle or 2.5 mg TGF-β3 24, 8, and 4 hours before irradiation and once immediately after irradiation. B) Animals were given vehicle or 2.5 mg IL-11 subcutaneously 1 day before irradiation, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm. C) Animals were given vehicle or 5 mg/kg KGF per day subcutaneously on days 2, 1, and 0; that is, before and at the time of (day 0) radiation. PB = partial body, WB = whole body, BMT = bone marrow transplant. Fig. 4. View largeDownload slide Each line represents the life of an animal through a survival experiment, with the black boxes indicating when diarrhea was observed and the termination of a line indicating when an animal died. This is taken from an experiment where a dose of 15.3 Gy partial body x-ray was delivered to the abdomen and the mice were pretreated with transforming growth factor β3 (top panel) or vehicle (lower panel). Fig. 4. View largeDownload slide Each line represents the life of an animal through a survival experiment, with the black boxes indicating when diarrhea was observed and the termination of a line indicating when an animal died. This is taken from an experiment where a dose of 15.3 Gy partial body x-ray was delivered to the abdomen and the mice were pretreated with transforming growth factor β3 (top panel) or vehicle (lower panel). References (1) Hendry JH, Roberts SA, Potten CS. The clonogen content of murine intestinal crypts: dependence on radiation dose used in its determination. Radiat Res  1992; 132: 115–9. Google Scholar (2) Cairnie AB, Millen BH. Fission of crypts in the small intestine of the irradiated mouse. Cell Tissue Kinet  1975; 8: 189–96. Google Scholar (3) Withers HR, Elkind M. Dose-survival characteristics of epithelial cells of mouse intestinal mucosa. Radiology  1968; 91: 998–1000. Google Scholar (4) Withers HR, Elkind M. Radiosensitivity and fractionation response of crypt cells of mouse jejunum. Radiat Res  1969; 38: 598–613. Google Scholar (5) Kurokawa M, Lynch K, Podolsky DK. Effects of growth factors on an intestinal epithelial cell line: transforming growth factor b inhibits proliferation and stimulates differentiation. Biochem Biophys Res Comm  1987; 142: 775–82. Google Scholar (6) Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc Natl Acad Sci U S A  1989; 86: 1578–82. Google Scholar (7) Withers HR, Elkind MM. Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int J Radiat Biol Relat Stud Phys Chem Med  1970; 7: 261–2. Google Scholar (8) Roberts SA. DRFIT: a program for fitting radiation survival models. Int J Radiat Biol Relat Stud Phys Chem Med  1990; 51: 1243–6. Google Scholar (9) Potten CS, Booth D, Haley JD. Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo. Br J Cancer  1997; 75: 1454–9. Google Scholar (10) Booth D, Haley JD, Bruskin AM, Potten CS. Transforming growth factor-b3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem cell cycling. Int J Cancer  2000; 86: 53–9 Google Scholar (11) Paul SR, Yang YC, Donahue RE, Goldring S, Williams DA. Stromal cell-associated haematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line. Blood  1991; 77: 1723–33. Google Scholar (12) Potten CS. Interleukin-11 protects the clonogenic stem cells in murine small-intestinal crypts from impairment of their reproductive capacity by radiation. Int J Cancer  1995; 62: 356–61. Google Scholar (13) Potten CS. Protection of the small intestinal clonogenic stem cells from radiation-induced damage by pretreatment with interleukin 11 also increases murine survival time. Stem Cells  1996; 14: 452–9. Google Scholar (14) Hiryama F, Shih J, Awgulewitsch A, Warr GW, Clark SC, Ogawa M. Clonal proliferation of murine lymphohemopoietic progenitors in culture. Proc Natl Acad Sci U S A  1992; 89: 5907–11. Google Scholar (15) Paul SR, Schendel P. The cloning and biological characterization of recombinant human interleukin 11. Int J Cell Cloning  1992; 10: 135–43. Google Scholar (16) Rubin JS, Osada H, Finch PW, Taylor WG, Rudikoff S, Aaronson SA. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci U S A  1989; 86: 802–6. Google Scholar (17) Housley RM, Morris CF, Boyle W, Ring B, Biltz R, Tarpley JE, et al. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J Clin Invest  1994; 94: 1764–77. Google Scholar (18) Farrell CL, Bready JV, Rex K, Chen JN, DiPalma CR, Whitcomb KL. et al Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality. Cancer Res  1998; 58: 933–39. Google Scholar (19) Farrell CL, Rex KL, Kaufman SA, Dipalma CR, Chen JN, Scully S. et al Effects of keratinocyte growth factor in the squamous epithelium of the upper aerodigestive tract of normal and irradiated mice. Int J Radiat Biol  1999; 75: 609–20. Google Scholar (20) Potten CS, O'Shea JA, Farrell CL, Rex K, Booth C. The effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine. Cell Growth Differ  2001; 12: 265–75. 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Protection Against Mucosal Injury By Growth Factors and Cytokines

JNCI Monographs , Volume 2001 (29) – Oct 1, 2001

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Abstract

Abstract This article provides an overview of published studies in which growth factors and cytokines were used to modify the sensitivity of intestinal stem cells to a dose of radiation. In these experiments, growth factors were used to manipulate the sensitivity of stem cells in the gastrointestinal tract to reduce the severity of gastrointestinal mucositis in cancer therapy patients. Transforming growth factor β3, interleukin 11, and keratinocyte growth factor were used. All three agents, given according to appropriate protocols, can result in a threefold to fourfold increase in the number of intestinal stem cells that survive a dose of radiation therapy. This result was assessed by using the crypt microcolony assay of stem cell functional capacity. The changes in stem cell survival that were observed resulted in increased animal survival. This increased survival was taken as a surrogate for improvement in patient well-being. The severity of diarrhea, a marker of functional impairment, was concomitantly reduced. Cancer therapy involves a fine balance between the use of a large enough dose of a drug to kill tumor cells and the prevention of damage to the normal tissues of the body, notably the oral mucosa, the gastrointestinal tract, and the hematopoietic system. Damage caused during cancer therapy to the epithelial layer lining the mouth causes the lining to become depleted and ulceration of the mouth to occur (oral mucositis). This makes it difficult for the patient to eat, swallow, or speak and causes pain and susceptibility to infection. A combination of patient discomfort and possible infection can lead to treatment delay or dose reduction, which can result in a less favorable outcome for the patient. Another major dose-limiting tissue is the gastrointestinal tract, whose rapid cell cycle makes it more susceptible to the effects of cytotoxic exposure and to a rapid expression of damage. This damage can be seen in the small intestine within a few days and within a slightly longer period of time in the large bowel. This article will concentrate on the specific problems associated with damage to the normal small intestine. The small intestine is a constantly renewing tissue, continuously replacing cells that are lost in the lumen of the intestine. This renewal is achieved by the production of new cells in the crypts by the stem cells and their progeny, arranged in an amplifying transit lineage of six to eight generations. Under steady- state situations, there are thought to be between four and 16 actual stem cells that are located near the base of the crypt. However, if the system is damaged (e.g., by radiation) an acute response occurs, causing some cells in the lower region of the crypt, possibly the stem cells, to die via apoptosis. The stem cells, probably along with some early transit generation cells, will recognize the damage and undergo rapid cell division to regenerate the crypt and, hence, the tissue by clonal growth. The number of clonogenic cells per crypt is thought to depend on the level of damage induced, but it could be as many as 30 cells per crypt (1). Some crypts will become reproductively sterile, will be unable to initiate this regeneration response, and will disappear within approximately 48 hours. The time sequence for this response is as follows (Fig. 1). In the first 2–3 days following a dose of radiation (e.g., a 14-Gy dose), some crypts can be seen to be regenerating (forming microcolonies) alongside other crypts that have been reproductively sterilized. On day 4 following radiation therapy, those crypts that have survived the radiation damage will be approximately 1.5 to two times bigger than a normal crypt, and the sterilized crypts will have disappeared. These large, regenerating crypts will then start to split or bud into several crypts (2). Approximately 14 days after irradiation, the regenerating crypts and foci will have grown visible to the naked eye as macrocolonies (3,4). In certain cases, budding will continue until the whole of the intestine has been regenerated and the normal architecture restored. There are advantages in protecting the clonogenic stem cells from damage, since they are the key to the survival of an individual crypt. The number of crypts that survive following cytotoxic damage determines how intact the intestinal mucosa is and, hence, how well an animal or a patient can survive the damage. The number of surviving crypts plays a pivotal role in the competitive race between depopulation and ulceration and regeneration. Alternatively, a stimulatory factor given before cytotoxic exposure could increase the number of stem cells per crypt that are subjected to the cytotoxic insult. A possible method of protecting the clonogenic stem cells might be to manipulate them by using growth factors. This could be achieved by using a growth factor that will take the stem cells out of the cycle, making them more resistant to the cytotoxic damage, and ensuring the survival of more stem cells capable of regenerating any damaged tissue. Finally, giving, after cytotoxic insult, a growth factor that could increase the rate of proliferation or initiate regeneration earlier may help in speeding up the regeneration process. A combination of various protocols could give maximum protection for the epithelium. Various experiments to manipulate clonogenic stem cells to protect against cytotoxic damage have been carried out, but this review will outline work that has used transforming growth factor β3 (TGF-β3), interleukin 11 (IL-11), and keratinocyte growth factor (KGF), using 10–12-week-old (C57BL/6 × DBA/2)F1 (BDF1) mice. All experiments were performed within the regulations of the U.K. Scientific Procedures Act (1986). Transforming Growth Factor β3 TGF-β3 is a known inhibitor of some epithelial cell proliferation (5,6) by preventing cell cycle progression and accumulating cells in G1 or G0. For clonogenic stem cells in the intestine, this might render them more resistant to the cytotoxic damage, leaving more clonogenic stem cells to start the regeneration process. To assess the capacity of TGF-β3 to protect the intestine from radiation damage, we used the crypt microcolony assay as described by Withers and Elkind (7). This is an accepted test for crypt stem cell functional capacity. A standard dose of 2.5 mg TGF-β3 was administered intraperitoneally to male BDF1 mice 24, 8, and 4 hours before and then once immediately after irradiation. Animals were culled and samples of small intestine were taken 4 days after irradiation. The number of surviving crypts per circumference (a unit length) was counted for 10 circumferences per mouse. Data were analysed by using the DRFIT program (8), which allows curves to be fitted and tests the statistical difference between treated and vehicle control groups by using a variance-ratio F test. As shown in Fig. 2, a, four injections of TGF-β3 administered once immediately after and again at 4, 8, and at 24 hours before radiation therapy compared with the vehicle control shows a statistically significant shift to the right of the treated curve (9) (P<.001). This indicates that there are four times more clonogenic stem cells surviving at 15 Gy in the TGF-β3-treated group than in the control group. It is interesting that if an additional dose of TGF-β3 was administered 4 hours after irradiation (–4, –8, –24, 0, and +4 hours), then the level of protection afforded is reduced (data not shown). TGF-β3 administered intraperitoneally after radiation therapy caused crypts to become sensitized, with only approximately one third of the crypts surviving, in comparison with the vehicle control (data not shown). This finding demonstrates the importance of the use of a growth factor in the correct protocol. These studies demonstrated that the small intestinal clonogenic stem cells can be protected from radiation damage. How does this finding relate, however, to the patient's well-being in cancer therapy? To answer this question, we conducted a series of experiments to study animal survival over time after a dose of radiation. Groups of 20 animals were pretreated with TGF-β3 or vehicle, then irradiated with a 15.8-Gy x-ray and observed for 30 days. Animals had their heads, thoraxes, and forelimbs shielded to reduce the complications of oral and hematopoietic damage. Almost 100% of animals pretreated with TGF-β3 survived to 30 days, but only approximately 35% of the vehicle control animals survived (10) (Fig. 3, a). During the 30 days postirradiation, animals were checked twice daily for diarrhea (verified by evidence of wet feces on the fur of the anal region). The duration of diarrhea can be seen on individual animal “lifelines” as shown in Fig. 4. Administration of TGF-β3 before the radiation dose reduces the total number of days during which diarrhea was recorded, suggesting not only an improvement in life expectancy within this experiment but also an improvement in quality of life and a reduction in diarrhea, a surrogate marker of intestinal dysfunction (10). The histology of the intestine of the animals surviving to 30 days is indistinguishable from that of control subjects (Fig. 1). This indicates truly remarkable regenerative capacity, especially when the appearance of the intestine at days 3–5 (Fig. 1) is considered. Interleukin 11 A pleiotrophic cytokine that affects many different systems, recombinant human IL-11 was originally isolated and cloned from an immortalized primate bone marrow stromal cell line (11). Similar experiments to the TGF-β3 study were carried out by using IL-11. A number of protocols were tested with use of the crypt microcolony assay, and these were carried out as follows: protocol A: IL-11 administered subcutaneously at 9 am and at 9 pm, for a total of five injections before irradiation; protocol B: IL-11 administered subcutaneously at 9 am and at 9 pm, for a total of seven injections postirradiation; and protocol C: IL-11 administered subcutaneously at 9 am and at 9 pm, for a total of three injections prior to irradiation and six injections following irradiation. In all cases, IL-11 protected the clonogenic stem cells from radiation damage, but to varying degrees. Protocol A gave good protection with a statistical significance of P<.001, and protocol B was only modestly protective (P = .003), while the best protection was afforded by administering IL-11 subcutaneously both before and after radiation therapy (protocol C; P<.001) (Fig. 2, b) (12), when up to 3.5 times more crypts survived a dose of 16-Gy irradiation in the IL-11 treated groups. Studies examining IL-11 and animal survival over time after radiation therapy did not take into consideration the damage caused to the hematopoietic system. Protocols A and C were carried out with a dose of 12-Gy x-ray irradiation. Animals were observed over the following 30 days. As a result of damage to the hematopoietic and gastrointestinal systems, all of the animals treated with a dose of 12-Gy irradiation died during the first 11 days; however, the IL-11-treated groups survived an average of 2 days longer than the controls (Fig. 3, b) (13). It is unclear by what mechanisms IL-11 protects against cytotoxic damage, but IL-11 given before radiation therapy alters the sensitivity of clonogenic stem cells, making them more resistant; hence, more of them survive. It is uncertain whether IL-11 alters cell cycle progression or has an indirect effect by altering other growth factors. IL-11 protects best when it is given both before and after irradiation, and it may act via different mechanisms before and after irradiation. IL-11 is known to synergize with other factors [e.g., stem cell factor and steel factor (14)] and also may enhance the effects of IL-3 (15). Keratinocyte Growth Factor KGF was identified originally as a factor that stimulated epithelial cells in vitro (16). Further studies demonstrated that it also stimulates both proliferation and differentiation in a number of epithelial cell types when given to healthy animals. This is very noticeable in the gastrointestinal tract (17). It has been observed that administration of KGF has trophic effects on the gastrointestinal tract, increasing crypt depth and villus length (18). KGF also has a particularly pronounced trophic effect on oral mucosa (19). Crypt survival studies with the use of KGF as a protective agent against radiation damage have shown that KGF given for 3 days before irradiation showed a statistically significant protective effect over a range of doses (P<.001) (Fig. 2, c), with three times more crypts surviving a dose of 16-Gy irradiation in the KGF treated group and 3.5 times more crypts surviving after a dose of 14-Gy irradiation than controls (vehicle treated) (18). Use of KGF postirradiation was not found to improve crypt survival. Animal survival studies have also been performed with KGF and 12 Gy of radiation by using postirradiation bone marrow transplantation to protect the hematopoietic system (Fig. 3, c). This experiment showed that 90% of mice pretreated with KGF before receiving 12 Gy of irradiation and a bone marrow transplant survived a 16-day observation period, whereas mice that received only 12 Gy of irradiation and a bone marrow transplant all died during the first 8 days. It seems somewhat puzzling that the best protocol for protection is to give KGF before cytotoxic insult when it is known that KGF is a stimulator. The mechanism by which KGF protects the intestinal system is unknown and could be multifactorial. However, KGF has been shown to have trophic effects on the gastrointestinal tract, increasing crypt size. This may suggest that KGF protects the crypts by proportionally increasing the number of clonogenic stem cells per crypt (18). Indeed, cell proliferation studies have clearly indicated increased stem cell proliferation following KGF treatment, as well as (rather surprisingly) a reduction in transit cell proliferation (20). Stimulatory factors, such as KGF and, possibly, IL-11, may also act as cell survival factors that prevent apoptosis. KGF has also been shown to increase the number of goblet cells in the gastrointestinal tract (17). Goblet cells produce mucins that act as a barrier between the epithelium and the luminal and trefoil proteins that act to defend the gut and can also aid in its repair. One difficulty in making comparisons between the effects of these different agents is that different injection regimens have been used, as have, in some cases, different radiation doses and delivery protocols. However, it is very clear that the sensitivity of the potential clonogenic stem cells in the small intestine of the mouse can be experimentally manipulated by exogenous growth factors or cytokines in an advantageous manner. Such manipulations have been shown to afford overall radioprotection to an animal, and this protection manifests itself in potentially dramatic changes in animal survival and well-being. Conclusions These studies show that statistically significant and, in some cases, dramatic reductions in mucositis can be effected by appropriate manipulation of stem cell sensitivity with the use of growth factors and cytokines. The results presented here are preliminary; extensive additional studies are required to determine the most effective doses and delivery protocols. Many more growth factors and cytokines should be tested, together with combined and sequential use of different factors. With the identification of intestinal-specific regulatory factors, it would seem likely that the sensitivity of the critical stem cells might be even more effectively manipulated. This would reduce the severity of gastrointestinal mucositis even further, improving the quality of life of cancer therapy patients and possibly allowing for dose escalation and improvement in cure rates. Fig. 1. View largeDownload slide Radiation effects on small intestine. (a) (Original magnification ×100 ). The earliest response is an increase in apoptosis 4.5 hours following 8-Gy irradiation. Arrows indicate apoptotic bodies. (b) (Original magnification ×50). A selected area of intestine demonstrating a single large regenerating crypt 4 days after 14-Gy irradiation. Vincristine helps identification by arresting cells in metaphase. Arrowheads = mitosis. Larger arrows indicate two reproductively sterilised crypts that are dying and disappearing. (c) (Original magnification ×40). Day 6 following 14-Gy irradiation. Many areas of intestine are completely devoid of crypts and villi with only a few epithelial cells remaining. (d) (Original magnification ×40.) Section through a normal (control) small intestine showing crypts and villi. (e) (Original magnification ×40). By day 8 following 14-Gy irradiation the few regenerating crypts (see Fig. 1, c) have split (budded) and are starting to form new crypts and villi. (f) (Original magnification ×40.) By day 30 following 14-Gy irradiation the epithelium in the surviving animals is restored to its normal small intestinal architecture, as seen in Fig. 1, d. Fig. 1. View largeDownload slide Radiation effects on small intestine. (a) (Original magnification ×100 ). The earliest response is an increase in apoptosis 4.5 hours following 8-Gy irradiation. Arrows indicate apoptotic bodies. (b) (Original magnification ×50). A selected area of intestine demonstrating a single large regenerating crypt 4 days after 14-Gy irradiation. Vincristine helps identification by arresting cells in metaphase. Arrowheads = mitosis. Larger arrows indicate two reproductively sterilised crypts that are dying and disappearing. (c) (Original magnification ×40). Day 6 following 14-Gy irradiation. Many areas of intestine are completely devoid of crypts and villi with only a few epithelial cells remaining. (d) (Original magnification ×40.) Section through a normal (control) small intestine showing crypts and villi. (e) (Original magnification ×40). By day 8 following 14-Gy irradiation the few regenerating crypts (see Fig. 1, c) have split (budded) and are starting to form new crypts and villi. (f) (Original magnification ×40.) By day 30 following 14-Gy irradiation the epithelium in the surviving animals is restored to its normal small intestinal architecture, as seen in Fig. 1, d. Fig. 2. View largeDownload slide Intestinal crypt survival curves comparing treated groups against respective vehicle controls. All vehicle groups are represented as open circles and all treated groups as closed symbols. (a) Animals were given vehicle or 2.5 mg transforming growth factor β3 (TGF-β3) intraperitoneally at 24, 8, and 4 hours before irradiation and once immediately after irradiation. Values defining the TGF-β3 survival curve are (mean ± SE) D0 = 1.26 ± 0.10, N = 5664 ± 4698 and the values for the vehicle curve are D0 = 1.12 ± 0.83, N = 5294 ± 4492. There is a statistically significant difference between the two curves (P<.001). D0 is the mean lethal dose, the reciprocal of the slope on the exponential position of the curve—it is a measure of the radiosensitivity; N is the back extrapolate to zero dose of the exponential portion of the curve and a measure of the size of the shoulder. (b) Animals were given vehicle or 2.5 mg interleukin 11 (IL-11) subcutaneously 1 day before, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm, for a total of three injections before irradiation and six injections after irradiation. Values for the IL-11 survival curves are (mean ± SE) D0 = 2.30 ± 0.94, N = 110.5 ± 23.7, and the values for the vehicle group are D0 = 1.84 ± 0.67, N = 232.5 ± 52.9. There is a significant difference between the two curves (P<.001). (c) Animals were given vehicle or 12.5 mg keratinocyte growth factor (KGF) subcutaneously once a day for 3 days before irradiation. Values for the KGF curve are D0 = 1.91 ± 0.12, and N = 173 ± 65, and the values for the vehicle control curve are D0 = 1.43 ± 0.06, and N = 886 ± 294. There is a statistically significant difference between the two curves (P<.001). Fig. 2. View largeDownload slide Intestinal crypt survival curves comparing treated groups against respective vehicle controls. All vehicle groups are represented as open circles and all treated groups as closed symbols. (a) Animals were given vehicle or 2.5 mg transforming growth factor β3 (TGF-β3) intraperitoneally at 24, 8, and 4 hours before irradiation and once immediately after irradiation. Values defining the TGF-β3 survival curve are (mean ± SE) D0 = 1.26 ± 0.10, N = 5664 ± 4698 and the values for the vehicle curve are D0 = 1.12 ± 0.83, N = 5294 ± 4492. There is a statistically significant difference between the two curves (P<.001). D0 is the mean lethal dose, the reciprocal of the slope on the exponential position of the curve—it is a measure of the radiosensitivity; N is the back extrapolate to zero dose of the exponential portion of the curve and a measure of the size of the shoulder. (b) Animals were given vehicle or 2.5 mg interleukin 11 (IL-11) subcutaneously 1 day before, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm, for a total of three injections before irradiation and six injections after irradiation. Values for the IL-11 survival curves are (mean ± SE) D0 = 2.30 ± 0.94, N = 110.5 ± 23.7, and the values for the vehicle group are D0 = 1.84 ± 0.67, N = 232.5 ± 52.9. There is a significant difference between the two curves (P<.001). (c) Animals were given vehicle or 12.5 mg keratinocyte growth factor (KGF) subcutaneously once a day for 3 days before irradiation. Values for the KGF curve are D0 = 1.91 ± 0.12, and N = 173 ± 65, and the values for the vehicle control curve are D0 = 1.43 ± 0.06, and N = 886 ± 294. There is a statistically significant difference between the two curves (P<.001). Fig. 3. View largeDownload slide Survival time of animals exposed to 12-Gy (experiment with interleukin-11 [IL-11]) or 15.8-Gy ( experiment with transforming growth factor β3[TGF-β3]) x-rays delivered whole body (IL-11) or abdomen only (TGF-β3). For the keratinocyte growth factor (KGF) experiment, 12-Gy Cs137 was delivered whole body, followed by a bone marrow transplant. All vehicle groups are represented as open symbols, and all treated groups are represented as closed symbols. A) Animals were given vehicle or 2.5 mg TGF-β3 24, 8, and 4 hours before irradiation and once immediately after irradiation. B) Animals were given vehicle or 2.5 mg IL-11 subcutaneously 1 day before irradiation, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm. C) Animals were given vehicle or 5 mg/kg KGF per day subcutaneously on days 2, 1, and 0; that is, before and at the time of (day 0) radiation. PB = partial body, WB = whole body, BMT = bone marrow transplant. Fig. 3. View largeDownload slide Survival time of animals exposed to 12-Gy (experiment with interleukin-11 [IL-11]) or 15.8-Gy ( experiment with transforming growth factor β3[TGF-β3]) x-rays delivered whole body (IL-11) or abdomen only (TGF-β3). For the keratinocyte growth factor (KGF) experiment, 12-Gy Cs137 was delivered whole body, followed by a bone marrow transplant. All vehicle groups are represented as open symbols, and all treated groups are represented as closed symbols. A) Animals were given vehicle or 2.5 mg TGF-β3 24, 8, and 4 hours before irradiation and once immediately after irradiation. B) Animals were given vehicle or 2.5 mg IL-11 subcutaneously 1 day before irradiation, at the time of irradiation, and continuously throughout the postirradiation regeneration phase. All injections were given at 9:00 am and 9:00 pm. C) Animals were given vehicle or 5 mg/kg KGF per day subcutaneously on days 2, 1, and 0; that is, before and at the time of (day 0) radiation. PB = partial body, WB = whole body, BMT = bone marrow transplant. Fig. 4. View largeDownload slide Each line represents the life of an animal through a survival experiment, with the black boxes indicating when diarrhea was observed and the termination of a line indicating when an animal died. This is taken from an experiment where a dose of 15.3 Gy partial body x-ray was delivered to the abdomen and the mice were pretreated with transforming growth factor β3 (top panel) or vehicle (lower panel). Fig. 4. View largeDownload slide Each line represents the life of an animal through a survival experiment, with the black boxes indicating when diarrhea was observed and the termination of a line indicating when an animal died. This is taken from an experiment where a dose of 15.3 Gy partial body x-ray was delivered to the abdomen and the mice were pretreated with transforming growth factor β3 (top panel) or vehicle (lower panel). References (1) Hendry JH, Roberts SA, Potten CS. 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Journal

JNCI MonographsOxford University Press

Published: Oct 1, 2001

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