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Transforming growth factors and intestinal epithelia: More questions than answers
GASTROENTEROLOGY
SELECTED
1989;97:1587-95
SUMMARIES
MARKFELDMAN, M.D.
Selected Summaries Editor Dallas Veterans Administration Medical Service (111) Dallas, Texas 75216
Medical Center
STAFF OF CONTRIBUTORS James L. Achord, Jackson, Miss. Eugene B. Chang, Chicago, Ill. Kiertisin Dharmsathaphorn, San Diego, Calif.
Charles 0. Elson, Birmingham, Ala. Hans Fromm, Washington, DC. Raymond S. Koff, Framingham, Mass. Robert C. Kurtz, New York, N.Y. M. Peter Lance, Buffalo, N.Y.
Arthur M. Magun, New York, N.Y. Peter F. Malet, Philadelphia, Pa. James McManus, Temple, Tex. Thomas A. Miller, Houston, Tex. Walter L. Peterson, Dallas, Tex. John H. C. Ranson, New York, N.Y. Joel E. Richter, Birmingham, Ala. Caroline A. Riely, Memphis, Tenn.
TRANSFORMING GROWTH FACTORS AND INTESTINAL EPITHELIA: MORE QUESTIONS THAN ANSWERS Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. (Departments of Cell Biology, Pediatrics, Surgery and Internal Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee) Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc Nat1 Acad Sci USA 1989;86:1578-82 (March). Koyama S, Podolsky DK (Departments of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts) Differential expression of transforming growth factors alpha and beta in rat intestinal epithelial cells. J Clin Invest 1989;83:1768-73 (May). The expression of transforming growth factors (TGF-(Y and TGF-/3) by two rat intestinal epithelial cell sources, i.e., primary cells and crypt-derived cell line (IEC-6), was examined in relation to states of cellular proliferation and differentiation. In the study by Koyama and Podolsky, TGF-P mRNA transcripts and activity were assessed in sequential fractions of epithelial cells isolated from villus to crypt regions of rat small intestine. Levels of TGF-/3l mRNA were found to be greatest in crypt cells and less in differentiated villus cells. Transforming growth factor-p activity, as determined by biological activity and radioreceptor binding inhibition assays, was distributed similarly along the villus-to-crypt axis. Unlike TGF-P expression, TGF-a (a factor known to stimulate cellular proliferation in a number of cell systems) appeared to be distributed oppositely, i.e., its activity and mRNA expression was greatest in the villus cells and least in crypt cells. Such a distribution of TGF-(Y and TGF-P was unexpected based on the known actions of these factors and in view of previous studies that demonstrated that TGF-P stimulation of intestinal epithelial IEC-6 cells inhibits proliferation and promotes cellular differentiation (Kurokawa and Podolsky, Biochem Biophys Res Commun 1987;42:775-82). In contrast to the findings of Koyama and Podolsky, those reported by Barnard et al. were significantly diver-
Konrad Schulze-Delrieu, Iowa City, Iowa Joseph Sweeting, New York, N.Y. Dwain L. Thiele, Dallas, Tex. Richard C. Thirlby, Seattle, Wash. Martin H. Ulshen, Chapel Hill, N.C. Ernest Urban, Pittsburgh, Pa.
gent. Transforming growth factor-@ activity and mRNA expression of TGF-P were greatest in mature villus cells and least in undifferentiated crypt cells of rat jejunum. Furthermore, TGF-P inhibited IEC-6 cell growth, but unlike previous reports by Kurokawa et al. (Biochem Biophys Res Commun 1987;142:775-82), failed to stimulate IEC-6 differentiation as assessed by sucrase and alkaline phosphatase activities. Transforming growth factor-p also increased in steady-state levels of its own mRNA within 8 h of treatment of rapidly growing IEC-6 cells. Comment. The intestinal mucosa is truly a unique tissue, for along its vertical axis the entire continuum of epithelial growth and differentiation can be witnessed, starting with undifferentiated progenitor cells in crypts and progressing to mature, multifunctional cells in villus. Exactly what factors underlay this orderly process of cellular differentiation has been a subject of intense interest, but one far from being unraveled. Largely from studies of their in vitro effects, a number of extracellular factors have recently been identified that could play a role in modulating intestinal mucosal growth and differentiation. Among these factors are extracellular matrix proteins (Am J Physiol 1988;254: G355-60). epidermal growth factor (Gastroenterology 1988;94: 65663), and TGF-a and TGF-p. Transforming growth factor-a is structurally homologous to EGF and has been shown to stimulate thymidine incorporation in many cell types (Cell BIochem 1986; 32:293-304, Nature 1987;328:817-20). In contrast, TGF-P has been found to inhibit cellular proliferation of most epithelial cells, although the cellular mechanism mediating its effects remains unknown. Recent studies by Kurokawa et al. (Biochem Biophys Res Commun 1987;142:775-82) have also suggested that TGF-/3 may induce differentiation of intestinal cultured IEC-6 cells, analogous to observations made in other tissues (J Cell Physiol 1987;133:426-37, Proc Nat1 Acad Sci USA 1986;83:243842, J Biol Chem 1986;261:16509-13). Expression of both TGF-a and TGF-P has also been shown in human colonic carcinomaderived cell lines (Cancer Res 1986;46:1164-9, Cancer Res 1987; 47:4590-4), keratinocytes (Cancer Res 1988;48:159&602), and, as these two studies now demonstrate, in normal primary and cultured intestinal cells. It is likely, therefore, that these factors have an important autocrine role. The study by Barnard et al., demonstrating expression and autoinduction of TGF-/3 in IEC-6 cells, provides further substantiation. The disagreement between these two studies regarding the
1588
GASTROENTEROLOGYVol. 97, No. 6
SELECTED SUMMARIES
differential expression of TGF-p along the crypt-to-villus axis underscores problems and the inherent limitations of current techniques used in this area of investigation. For instance, differences in the way cells are isolated from villus to crypt could account for varying degrees of contamination by nonepithelial lamina propria cells. Regional differences may exist along the longitudinal axis of the intestine such that the distribution of TGFs may differ significantly between the proximal and distal areas of the intestine. Identification of TGF-a and TGF-p by biological or radioreceptor binding inhibition assays may be insufficient to positively localize these proteins. Finally, the lack of adequate normalizing factors for comparison of steady-state levels of mRNA in villus and crypt extracts could account for artifact. For example, normalization of mRNA level of TGF-P to that of cyclophyllin, as was done by Barnard et al., could be misleading if the latter is also differentially expressed along the villus-to-crypt axis as suggested by Koyama and Podolsky. Unfortunately, neither study helps us in understanding the function and role of TGF-(Yor TGF-/3 in intestinal mucosa. “Guilt by association” is clearly not enough. Perhaps the most important lesson learned from these studies is that our current conceptual framework of how TGFs work may be insufficient. The actions of TGF-(r and TGF-p may be much more complex than one stimulating proliferation and the other inducing maturation. Perhaps both factors work in concert to induce or modulate cellular function or differentiation. Conceivably, cellular responses to these factors may differ depending on their state of differentiation. Possibly, TGF-a or TGF-P may have a role outside of direct regulation of growth and differentiation that ultimately may or may not impact on cellular function (J Cell Biol 1987;105:103945). Clearly we have only a few pieces to this exciting puzzle. E. B. CHANG, M.D.
Reply. Since publication of our paper, additional studies have further supported the finding of maximal TGF-Pl expression in the terminally differentiated villus tip cell. In situ hybridization studies of mouse TGF-/31 distribution in the jejunum clearly shows maximal expression in the villus tip (unpublished observations). It is important to appreciate that the signal is seen not only in epithelial cells, but also in the underlying lamina propria. Wakefield and coworkers have shown TGF-/31 protein in the villus tip of the small intestine by immunohistochemical staining (NY Acad Sci Ann 1988;551:290). Little or no staining was found in the crypt. As pointed out in our paper, the aforementioned distribution of TGF-/ll is analogous to that described in another major epithelial surface, the skin. J. A.
BARNARD
III, M.D.
MORE BANG FOR THE BUCK IN BANGKOK: USING HEPATITIS B VIRUS VACCINE WITHOUT HEPATITIS B IMMUNE GLOBULIN Poovorawan Y, Sanpavat S, Pongpuniert W, et al. (Departments of Pediatrics and Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University and Hospital, Bangkok, Thailand, and Medical and Scientific Services, SmithKline Biologicals, Rixensart, Belgium) Protective efficacy of a recombinant DNA hepatitis B vaccine in neonates of HBe antigen-positive mothers. JAMA 1989;261: 3278-81 (June 9).
For neonates of women in the United States identified as hepatitis B surface antigen (HBsAG) carriers, regardless of hepatitis B e antigen (HBeAg) status, interruption of maternal-neonatal infection by immunization with hepatitis B virus (HBV) vaccine [active immunization) together with hepatitis B immune globulin (HBIG) (passive immunization) has been strongly recommended. Early studies with plasma-derived vaccines suggested that combined activepassive immunization was more efficacious than active or passive immunization alone. Combined active-passive immunization also has been shown to provide longer protection than passive immunization alone. But combined immunization programs are also more costly than the use of a single approach. The introduction of the first yeastderived recombinant HBV vaccine, just a few years ago, did not lead to reduction in the cost of active immunization. For countries in which maternal-neonatal transmission is common and economic resources are limited, active immunization alone [if shown to be equivalent in effectiveness to combined immunization) would be advantageous since it would be cheaper. In this report from Thailand the protective efficacy of active immunization alone was studied in the neonates of, HBsAg- and HBeAg-positive mothers. Measurement of serum levels of HBV DNA in these women indicated that the majority (71%) had levels above 9.95 pg/pl, a level considered consistent with high infectivity. A yeast-derived recombinant DNA vaccine (Engerix-B), produced by SmithKline Biologicals, Rixensart, Belgium, was given to neonates in a dose of 10 pg, into the anterolateral muscle of the thigh, within 12 h of birth. Subsequent doses were given at 1, 2, and 12 mo of age. Hepatitis B immune globulin was not given and no placebo-treated control group was studied. Among 55 identified neonates who completed the four-dose vaccination schedule, and who were followed for at least 13 mo, only 5 had positive tests for HBsAg. In 3 of these, HBsAg was present at birth, but later samples were negative. One infant was HBsAg-positive at birth and remained so, and one became positive at 1 mo of age and stayed positive during follow-up. Thus 2 of the 55 infants have become HBsAg carriers. The authors estimate the protective efficacy rate of the yeast-derived recombinant DNA vaccine used in this study to be 94.5%, assuming a conservative neonatal persistent infection rate of 65% if no immunization had been given. Comment. This study is of considerable importance because it is consistent with several other recent studies suggesting that HBIG may not be necessary in the setting of postexposure immunoprophylaxis for maternal-neonatal transmission, even if high-risk neonates are the study population. In a pilot study undertaken in China, protective efficacy rates were not increased by the addition of HBIG to a low-dose vaccination program (J Cell Physiol [Suppl] 1986;4:83-90). In Indonesia, only 3 (11%) of 27 infants of HBsAgpositive women given the same yeast-recombinant DNA vaccine as used in Thailand, in a four-dose schedule [within 24 h of birth, and at 1,2, and 12 mo], without HBIG, became HBsAg carriers (In: Zuckerman A, ed. Viral hepatitis and Jiver disease, New York: Alan Liss, 1988:1059-61). Although it is unfortunate that active immunization alone was not directly compared with combined active-passive immunization in the Thailand study, it would be difficult to demonstrate a
SELECTED
1989;97:1587-95
SUMMARIES
MARKFELDMAN, M.D.
Selected Summaries Editor Dallas Veterans Administration Medical Service (111) Dallas, Texas 75216
Medical Center
STAFF OF CONTRIBUTORS James L. Achord, Jackson, Miss. Eugene B. Chang, Chicago, Ill. Kiertisin Dharmsathaphorn, San Diego, Calif.
Charles 0. Elson, Birmingham, Ala. Hans Fromm, Washington, DC. Raymond S. Koff, Framingham, Mass. Robert C. Kurtz, New York, N.Y. M. Peter Lance, Buffalo, N.Y.
Arthur M. Magun, New York, N.Y. Peter F. Malet, Philadelphia, Pa. James McManus, Temple, Tex. Thomas A. Miller, Houston, Tex. Walter L. Peterson, Dallas, Tex. John H. C. Ranson, New York, N.Y. Joel E. Richter, Birmingham, Ala. Caroline A. Riely, Memphis, Tenn.
TRANSFORMING GROWTH FACTORS AND INTESTINAL EPITHELIA: MORE QUESTIONS THAN ANSWERS Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. (Departments of Cell Biology, Pediatrics, Surgery and Internal Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee) Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc Nat1 Acad Sci USA 1989;86:1578-82 (March). Koyama S, Podolsky DK (Departments of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts) Differential expression of transforming growth factors alpha and beta in rat intestinal epithelial cells. J Clin Invest 1989;83:1768-73 (May). The expression of transforming growth factors (TGF-(Y and TGF-/3) by two rat intestinal epithelial cell sources, i.e., primary cells and crypt-derived cell line (IEC-6), was examined in relation to states of cellular proliferation and differentiation. In the study by Koyama and Podolsky, TGF-P mRNA transcripts and activity were assessed in sequential fractions of epithelial cells isolated from villus to crypt regions of rat small intestine. Levels of TGF-/3l mRNA were found to be greatest in crypt cells and less in differentiated villus cells. Transforming growth factor-p activity, as determined by biological activity and radioreceptor binding inhibition assays, was distributed similarly along the villus-to-crypt axis. Unlike TGF-P expression, TGF-a (a factor known to stimulate cellular proliferation in a number of cell systems) appeared to be distributed oppositely, i.e., its activity and mRNA expression was greatest in the villus cells and least in crypt cells. Such a distribution of TGF-(Y and TGF-P was unexpected based on the known actions of these factors and in view of previous studies that demonstrated that TGF-P stimulation of intestinal epithelial IEC-6 cells inhibits proliferation and promotes cellular differentiation (Kurokawa and Podolsky, Biochem Biophys Res Commun 1987;42:775-82). In contrast to the findings of Koyama and Podolsky, those reported by Barnard et al. were significantly diver-
Konrad Schulze-Delrieu, Iowa City, Iowa Joseph Sweeting, New York, N.Y. Dwain L. Thiele, Dallas, Tex. Richard C. Thirlby, Seattle, Wash. Martin H. Ulshen, Chapel Hill, N.C. Ernest Urban, Pittsburgh, Pa.
gent. Transforming growth factor-@ activity and mRNA expression of TGF-P were greatest in mature villus cells and least in undifferentiated crypt cells of rat jejunum. Furthermore, TGF-P inhibited IEC-6 cell growth, but unlike previous reports by Kurokawa et al. (Biochem Biophys Res Commun 1987;142:775-82), failed to stimulate IEC-6 differentiation as assessed by sucrase and alkaline phosphatase activities. Transforming growth factor-p also increased in steady-state levels of its own mRNA within 8 h of treatment of rapidly growing IEC-6 cells. Comment. The intestinal mucosa is truly a unique tissue, for along its vertical axis the entire continuum of epithelial growth and differentiation can be witnessed, starting with undifferentiated progenitor cells in crypts and progressing to mature, multifunctional cells in villus. Exactly what factors underlay this orderly process of cellular differentiation has been a subject of intense interest, but one far from being unraveled. Largely from studies of their in vitro effects, a number of extracellular factors have recently been identified that could play a role in modulating intestinal mucosal growth and differentiation. Among these factors are extracellular matrix proteins (Am J Physiol 1988;254: G355-60). epidermal growth factor (Gastroenterology 1988;94: 65663), and TGF-a and TGF-p. Transforming growth factor-a is structurally homologous to EGF and has been shown to stimulate thymidine incorporation in many cell types (Cell BIochem 1986; 32:293-304, Nature 1987;328:817-20). In contrast, TGF-P has been found to inhibit cellular proliferation of most epithelial cells, although the cellular mechanism mediating its effects remains unknown. Recent studies by Kurokawa et al. (Biochem Biophys Res Commun 1987;142:775-82) have also suggested that TGF-/3 may induce differentiation of intestinal cultured IEC-6 cells, analogous to observations made in other tissues (J Cell Physiol 1987;133:426-37, Proc Nat1 Acad Sci USA 1986;83:243842, J Biol Chem 1986;261:16509-13). Expression of both TGF-a and TGF-P has also been shown in human colonic carcinomaderived cell lines (Cancer Res 1986;46:1164-9, Cancer Res 1987; 47:4590-4), keratinocytes (Cancer Res 1988;48:159&602), and, as these two studies now demonstrate, in normal primary and cultured intestinal cells. It is likely, therefore, that these factors have an important autocrine role. The study by Barnard et al., demonstrating expression and autoinduction of TGF-/3 in IEC-6 cells, provides further substantiation. The disagreement between these two studies regarding the
1588
GASTROENTEROLOGYVol. 97, No. 6
SELECTED SUMMARIES
differential expression of TGF-p along the crypt-to-villus axis underscores problems and the inherent limitations of current techniques used in this area of investigation. For instance, differences in the way cells are isolated from villus to crypt could account for varying degrees of contamination by nonepithelial lamina propria cells. Regional differences may exist along the longitudinal axis of the intestine such that the distribution of TGFs may differ significantly between the proximal and distal areas of the intestine. Identification of TGF-a and TGF-p by biological or radioreceptor binding inhibition assays may be insufficient to positively localize these proteins. Finally, the lack of adequate normalizing factors for comparison of steady-state levels of mRNA in villus and crypt extracts could account for artifact. For example, normalization of mRNA level of TGF-P to that of cyclophyllin, as was done by Barnard et al., could be misleading if the latter is also differentially expressed along the villus-to-crypt axis as suggested by Koyama and Podolsky. Unfortunately, neither study helps us in understanding the function and role of TGF-(Yor TGF-/3 in intestinal mucosa. “Guilt by association” is clearly not enough. Perhaps the most important lesson learned from these studies is that our current conceptual framework of how TGFs work may be insufficient. The actions of TGF-(r and TGF-p may be much more complex than one stimulating proliferation and the other inducing maturation. Perhaps both factors work in concert to induce or modulate cellular function or differentiation. Conceivably, cellular responses to these factors may differ depending on their state of differentiation. Possibly, TGF-a or TGF-P may have a role outside of direct regulation of growth and differentiation that ultimately may or may not impact on cellular function (J Cell Biol 1987;105:103945). Clearly we have only a few pieces to this exciting puzzle. E. B. CHANG, M.D.
Reply. Since publication of our paper, additional studies have further supported the finding of maximal TGF-Pl expression in the terminally differentiated villus tip cell. In situ hybridization studies of mouse TGF-/31 distribution in the jejunum clearly shows maximal expression in the villus tip (unpublished observations). It is important to appreciate that the signal is seen not only in epithelial cells, but also in the underlying lamina propria. Wakefield and coworkers have shown TGF-/31 protein in the villus tip of the small intestine by immunohistochemical staining (NY Acad Sci Ann 1988;551:290). Little or no staining was found in the crypt. As pointed out in our paper, the aforementioned distribution of TGF-/ll is analogous to that described in another major epithelial surface, the skin. J. A.
BARNARD
III, M.D.
MORE BANG FOR THE BUCK IN BANGKOK: USING HEPATITIS B VIRUS VACCINE WITHOUT HEPATITIS B IMMUNE GLOBULIN Poovorawan Y, Sanpavat S, Pongpuniert W, et al. (Departments of Pediatrics and Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University and Hospital, Bangkok, Thailand, and Medical and Scientific Services, SmithKline Biologicals, Rixensart, Belgium) Protective efficacy of a recombinant DNA hepatitis B vaccine in neonates of HBe antigen-positive mothers. JAMA 1989;261: 3278-81 (June 9).
For neonates of women in the United States identified as hepatitis B surface antigen (HBsAG) carriers, regardless of hepatitis B e antigen (HBeAg) status, interruption of maternal-neonatal infection by immunization with hepatitis B virus (HBV) vaccine [active immunization) together with hepatitis B immune globulin (HBIG) (passive immunization) has been strongly recommended. Early studies with plasma-derived vaccines suggested that combined activepassive immunization was more efficacious than active or passive immunization alone. Combined active-passive immunization also has been shown to provide longer protection than passive immunization alone. But combined immunization programs are also more costly than the use of a single approach. The introduction of the first yeastderived recombinant HBV vaccine, just a few years ago, did not lead to reduction in the cost of active immunization. For countries in which maternal-neonatal transmission is common and economic resources are limited, active immunization alone [if shown to be equivalent in effectiveness to combined immunization) would be advantageous since it would be cheaper. In this report from Thailand the protective efficacy of active immunization alone was studied in the neonates of, HBsAg- and HBeAg-positive mothers. Measurement of serum levels of HBV DNA in these women indicated that the majority (71%) had levels above 9.95 pg/pl, a level considered consistent with high infectivity. A yeast-derived recombinant DNA vaccine (Engerix-B), produced by SmithKline Biologicals, Rixensart, Belgium, was given to neonates in a dose of 10 pg, into the anterolateral muscle of the thigh, within 12 h of birth. Subsequent doses were given at 1, 2, and 12 mo of age. Hepatitis B immune globulin was not given and no placebo-treated control group was studied. Among 55 identified neonates who completed the four-dose vaccination schedule, and who were followed for at least 13 mo, only 5 had positive tests for HBsAg. In 3 of these, HBsAg was present at birth, but later samples were negative. One infant was HBsAg-positive at birth and remained so, and one became positive at 1 mo of age and stayed positive during follow-up. Thus 2 of the 55 infants have become HBsAg carriers. The authors estimate the protective efficacy rate of the yeast-derived recombinant DNA vaccine used in this study to be 94.5%, assuming a conservative neonatal persistent infection rate of 65% if no immunization had been given. Comment. This study is of considerable importance because it is consistent with several other recent studies suggesting that HBIG may not be necessary in the setting of postexposure immunoprophylaxis for maternal-neonatal transmission, even if high-risk neonates are the study population. In a pilot study undertaken in China, protective efficacy rates were not increased by the addition of HBIG to a low-dose vaccination program (J Cell Physiol [Suppl] 1986;4:83-90). In Indonesia, only 3 (11%) of 27 infants of HBsAgpositive women given the same yeast-recombinant DNA vaccine as used in Thailand, in a four-dose schedule [within 24 h of birth, and at 1,2, and 12 mo], without HBIG, became HBsAg carriers (In: Zuckerman A, ed. Viral hepatitis and Jiver disease, New York: Alan Liss, 1988:1059-61). Although it is unfortunate that active immunization alone was not directly compared with combined active-passive immunization in the Thailand study, it would be difficult to demonstrate a