Changes in histology and fibrogenic cytokines in irradiated colorectum of two murine strains

doi:10.1016/S0360-3016(98)00201-6

International Journal of Radiation OncologyBiologyPhysics

Volume 42, Issue 1, 1 August 1998, Pages 169-178

https://doi.org/10.1016/S0360-3016(98)00201-6 Get rights and content

Abstract

Purpose: A strain difference in the development of radiation-induced fibrosis of the colorectum was recently observed. C57B1/6 mice developed colorectal obstruction with significantly higher incidence compared to C3Hf/Kam mice after partial volume irradiation with 30 Gy. Previous reports have demonstrated differences in cytokine mRNA levels in fibrosis-prone and -resistant mice after lung irradiation. The aims of this study are to determine if there are strain differences in: 1) the histology of the lesion, 2) mRNA levels for transforming growth factor beta (TGFβ) isoforms and tumor necrosis factor alpha (TNFα), and 3) immunohistochemical staining patterns using antibodies against the TGFβ isoforms and latency-associated peptide (LAP).

Methods and Materials: The colorectum of male C3Hf/Kam (C3H) and C57Bl/6 (B6) mice were irradiated using a dose/length combination (30 Gy to 13.7 mm) that resulted in 10 or 100% incidence of obstruction by 6 months in each strain, respectively. Colorectal tissue was removed from 6 hours to 120 days after irradiation as well as from obstructed mice and prepared for histology, RNase protection assay, and immunofluorescence.

Results: Distinct differences in the histological phenotype for the two strains were observed at times preceding obstruction. Samples from B6 mice showed increased hyperplastic crypts, colitis cystica profunda, and fibrosis within the lamina propria, compared to identically treated C3H mice. Fibrosis in the lamina propria of B6 mice appeared early, beginning at 75 days after irradiation, and was progressive, whereas fibrosis in C3H mice appeared simultaneous with obstruction and may have been a reaction to ulceration. No consistent strain difference in mRNA levels for TGFβ1, 2, 3 or TNFα were observed, although mRNA levels of TGFβ1 and TNFα were significantly elevated in both strains relative to nonirradiated controls. Immunofluorescent staining for TGFβ1, 3 and LAP was observed in hyperplastic crypts and colitis cystica profunda adjacent to regions of fibrosis, histological changes that were present predominately in the B6 strain.

Conclusions: The response of the colorectum to irradiation involves changes in the expression of several different cytokines. However, the lack of a consistent strain difference in TGFβ1, 2, 3 and TNFα mRNA levels, despite strain differences in both the incidence of colorectal obstruction and histological features preceding obstruction, suggests that mRNA changes in these fibrogenic cytokines are not the critical determinant of the strain difference and are not related to the process of radiation-induced colorectal fibrosis in these mouse strains. Strain-dependent differences were observed in the localization of active TGFβ, but these differences were related to the histological changes specifically found in the irradiated colon of the B6 strain.

Introduction

It has been suggested that some late effects in irradiated normal tissues result from a dynamic and complex interaction among many different cell types within a tissue that communicate via release of various cytokines with roles in promoting connective tissue accumulation leading to fibrosis 1, 2, 3. The balance among various cytokines, which may have opposing effects, as well as dysregulation of their levels may be important in the development of certain chronic radiation responses 1, 3, 4. Epithelial and inflammatory cells are sources of cytokines that stimulate fibroblasts to produce extracellular matrix proteins 5, 6. Transforming growth factor beta (TGFβ) is an important cytokine that may both initiate and terminate tissue repair after injury, but whose unregulated production may underlie the development of tissue fibrosis (7). TGFβ1 is known to stimulate processes that enhance the accumulation of extracellular matrix (ECM) components through several mechanisms, including the chemoattraction of fibroblasts and macrophages 7, 8, the upregulation of ECM component synthesis by fibroblasts and intestinal smooth muscle cells 9, 10, the downregulation of proteases (11), the upregulation of protease inhibitors 11, 12, and the upregulation of matrix adhesion molecules (13). TGFβ is thought to have differential effects on the reepithelialization of intestinal epithelium after injury since it both inhibits epithelial cell proliferation and stimulates the migration of epithelial cells into a wounded surface, in vitro 14, 15, 16. There is evidence that the isoforms of TGFβ may have different roles in the wound healing process (17). Although TGFβ1 may promote fibrosis, TGFβ3 has been shown to reduce connective tissue formation and subsequent scarring after wounding in rats 18, 19. The balance between TGFβ1, TGFβ3 and their receptors may be important factors in determining if a wound heals with or without scarring 17, 18, 19.

Alteration of TGFβ levels has been observed after irradiation of several normal tissues including skin 20, 21, liver 22, 23, bladder (24), mammary gland 25, 26, lung 3, 5, 6, and intestine 2, 27, 28. Strain differences in TGFβ have been observed with higher levels in fibrosis-prone mouse strains (C57Bl/6 or C57L/J) compared to fibrosis-resistant mouse strains (C3H/HeJ or C3HeB/FeJ) after lung irradiation 3, 6.

Other possible participants in fibrogenesis include proinflammatory cytokines, such as tumor necrosis factor alpha (TNFα), which trigger the recruitment of inflammatory cells through the expression of adhesion molecules on the vascular surface as well as the stimulation of fibroblast proliferation 29, 30, 31. Alteration of TNFα levels has been observed after irradiation of lung (32) or brain 33, 34 and in pulmonary fibrosis induced by bleomycin or silica 35, 36, 37. A strain difference in TNFα mRNA has been observed with higher levels in the fibrosis-prone C57Bl/6 strain, compared to the fibrosis-resistant C3H/HeJ strain, at early times after lung irradiation (32).

In a recent study, C57Bl/6 mice were observed to develop radiation-induced colorectal obstruction at significantly higher incidence compared to identically treated C3Hf/Kam mice (38). Strain differences in fibrogenic cytokine levels after colorectal irradiation have not been previously reported. We hypothesized that differences in the levels of the fibrogenic cytokines TGFβ and TNFα contribute to the higher incidence of colorectal obstruction observed after irradiation of C57Bl/6 mice compared to C3Hf/Kam mice. The aims of the current study are to determine if there are strain differences after irradiation of C57Bl/6 and C3Hf/Kam mouse colorectum in: 1) histological changes in the development of the fibrotic lesion, 2) the immunohistochemical staining pattern using antibodies against the TGFβ isoforms and latency-associated peptide (LAP), and 3) mRNA levels for the TGFβ isoforms and TNFα.

Section snippets

Animals

Male C3Hf/Kam (C3H) and C57Bl/6 (B6) mice weighing 27 to 33 g and 4 to 6 months old were bred and maintained in our specific pathogen–free animal colony. The animal facility is approved by the American Association for Accreditation of Laboratory Animal Care, and the animal care program operates in accordance with current U.S. Department of Agriculture, Department of Health and Human Services, and National Institutes of Health regulations and standards.

Irradiation

Details of the irradiation procedure have

Histology

Histological sections obtained at 15-day intervals up to 120 days after 30 Gy to 13.7 mm of colorectum were examined to provide a qualitative description of strain differences in reepithelialization postirradiation and in the development of the fibrotic lesion associated with obstruction (Table 1). At day 15, the epithelium from both B6 and C3H mice was denuded, although beginning to regenerate in a manner consistent with previous observations (43). After this time, however, distinct

Discussion

Recently, a significant difference in the incidence of radiation-induced colorectal obstruction was observed for the two mouse strains used in this study. The C57Bl/6 (B6) mice had higher incidence (100%) compared to the C3Hf/Kam (C3H) mice (10%) by 6 months after irradiation of 13.7 mm of colorectum with 30 Gy (38). The latency time for obstruction was also strain-dependent (i.e. B6—93 days, C3H—124 days). This observation was consistent with previous reports showing that different B6 strains

Acknowledgements

The authors thank Dr. Mary Helen Barcellos-Hoff and Shraddha Ravani for their generous gift of time and training regarding the immunohistochemical staining technique. We also thank Abraham Kuriakose for histological staining, Dr. Daniel Carson and JoAnne Julian for use of the cryostat, Dr. Susan Tucker for advice on statistics, Dr. Walter Hittleman for helpful discussions, Bati Bickerstaff and Patricia Skwarchuk for help in preparation of this manuscript.

References (56)

P Rubin et al.
A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis
Int J Radiat Oncol Biol Phys
(1995)
J.N Finkelstein et al.
Early alterations in extracellular matrix and transforming growth factor β gene expression in mouse lung indicative of late radiation fibrosis
Int J Radiat Oncol Biol Phys
(1994)
H.P Rodemann et al.
Cellular basis of radiation-induced fibrosis
Radiother Oncol
(1995)
R.A Ignotz et al.
Transforming growth factor-β stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix
J Biol Chem
(1986)
M.F Graham et al.
Transforming growth factor beta 1 selectively augments collagen synthesis by human intestinal smooth muscle cells
Gastroenterol
(1990)
M Laiho et al.
Transforming growth factor beta induction of type 1 plasminogen activator inhibitor
J Biol Chem
(1987)
R.A Ignotz et al.
Cell adhesion protein receptors as targets for transforming growth factor beta action
Cell
(1987)
A.U Dignass et al.
Cytokine modulation of intestinal epithelial cell restitutionCentral role of transforming growth factor β
Gastroenterol
(1993)
M Göke et al.
Regulation of the mucosal epithelial barrier
Bailliere’s Clin Gastroenterol
(1996)
S Frank et al.
Transforming growth factors β1, β2 and β3 and their receptors are differentially regulated during normal and impaired wound healing
J Biol Chem
(1996)

S O’Kane et al.

Transforming growth factor betas and wound healing

Int J Biochem Cell Biol

(1997)

C.W Langberg et al.

Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation

Radiother Oncol

(1994)

K.K Richter et al.

Association of transforming growth factor beta (TGF-beta) immunoreactivity with specific histopathologic lesions in subacute and chronic experimental radiation enteropathy

Radiother Oncol

(1996)

J.-H Hong et al.

Induction of acute phase gene expression by brain irradiation

Int J Radiat Oncol Biol Phys

(1995)

P.F Piguet

Cytokines involved in pulmonary fibrosis

Int Rev Exp Pathol

(1993)

M.W Skwarchuk et al.

Strain-dependent differences in the incidence of fibrotic obstruction and the volume effect after irradiation of mouse colorectum

Int J Radiat Oncol Biol Phys

(1998)

P Chomczynski et al.

Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction

Anal Biochem

(1987)

R.K Kumar et al.

Messages and handshakesCellular interactions in pulmonary fibrosis

Pathology

(1995)

P Rubin et al.

Molecular biology mechanisms in the radiation induction of pulmonary injury syndromesInterrelationship between the alveolar macrophage and the septal fibroblast

Int J Radiat Oncol Biol Phys

(1992)

K.R Trott et al.

Experimental studies on the pathogenesis of the chronic radiation ulcer of the large bowel in rats

Int J Radiat Oncol Biol Phys

(1986)

P.A Canney et al.

Transforming growth factor betaA promotor of late connective tissue injury following radiotherapy?

Br J Radiol

(1990)

A.J Franko et al.

Immunohistochemical localization of transforming growth factor beta and tumor necrosis factor alpha in the lungs of fibrosis-prone and “non-fibrosing” mice during the latent period and early phase after irradiation

Radiat Res

(1997)

J Wang et al.

Radiation-induced alteration of rat mesangial cell transforming growth factor-β and expression of the genes associated with the extracellular matrix

Radiat Res

(1996)

C.J Johnston et al.

Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis

Radiat Res

(1995)

W.A Border et al.

Transforming growth factor beta in tissue fibrosis

N Engl J Med

(1994)

D.R Edwards et al.

Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor

EMBO J

(1987)

J.A Barnard et al.

Regulation of intestinal epithelial cell growth by transforming growth factor type beta

Proc Natl Acad Sci USA

(1989)

M Shah et al.

Neutralisation of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring

J Cell Sci

(1995)

Cited by (51)

Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis
2015, American Journal of Pathology
Citation Excerpt :
Animals were anesthetized (1.5% isoflurane), and a single dose of 27-Gy γ irradiation (1.4 Gy/minute) was delivered by a cobalt 60 source through a 1 × 0.8-cm window centered on the colorectal region. This model of localized single-dose radiation exposure does not simulate fractionation treatment, but generates histopathological lesions similar to those seen clinically (ie, severe acute mucosal ulceration and transmural collagen deposition during the late phase), with 100% rectal obstruction, as previously reported.27–29 At different times after radiation exposure, colorectal tissues were removed and paraffin sections (5 μm thick) were stained with hematoxylin-eosin-saffron for routine histological examination.
The endothelial-to-mesenchymal transition (EndoMT) is a crucial cellular process during heart development necessary to the formation of cardiac valves. This embryonic process reappears in several pathological situations, such as vascular injury or organ fibrosis of various etiologies, as a mediator of extracellular matrix-producing cells. Because radiation induces both vascular damage and fibrosis, we investigated whether radiation exposure induces EndoMT in primary human intestinal microvascular endothelial cells (HIMECs) and whether EndoMT contributes to radiation-induced rectal damage in humans and in a preclinical model of radiation proctitis in mice. Irradiated HIMECs show phenotypic hallmarks of radiation-induced endothelial cell activation in vitro. Moreover, HIMECs undergo changes in molecular expression pattern compatible with EndoMT, with up-regulation of mesenchymal markers and down-regulation of endothelial markers via transforming growth factor/Smad pathway activation. In vivo, EndoMT readily occurs in the human rectum after radiation therapy for rectal adenocarcinoma. Finally, EndoMT was observed in rectal mucosal and submucosal microvessels in a preclinical model of radiation proctitis in Tie2-green fluorescent protein reporter–expressing mice all along radiation proctitis development, also associated with transforming growth factor/Smad pathway activation. In conclusion, radiation-induced cell activation and tissue inflammation constitute a setting that fosters the phenotypic conversion of endothelial cells into mesenchymal cells. Therefore, EndoMT is identified as a potential participant in radiation-induced gut damage and may represent an interesting therapeutic target in cases of radiation-induced pelvic disease.
Gastrointestinal complications of pelvic radiotherapy: Medical and surgical management strategies
2013, Current Problems in Surgery
Citation Excerpt :
During treatment with radical external-beam radiotherapy, an acute inflammatory reaction is seen in exposed parts of the intestine. Data regarding changes in small bowel during this phase are limited to animal studies.6 Caution must be exercised in extrapolating these findings to humans, however, because animals in these studies frequently receive fractionated regimens unlikely to be analogous to those employed in humans and a uniform response to a given radiation dose is unlikely to exist in different animal models.7
Clinical experience in 89 consecutive cases of chronic radiation enterocolitis
2011, Journal of the Chinese Medical Association
Citation Excerpt :
In our series, we confirmed smoking as an independent risk factor associated with surgery. Recent human and animal studies suggest that elevated levels of mucosal proinflammatory cytokines such as interleukin 2, interleukin 6 and tumor-necrosis factor α may be responsible for vulnerability to radiation enterocolitis.20,21 It is likely that smoking has an effect on systemic inflammatory response and hence on elevated proinflammatory cytokines, making patients more vulnerable to severe radiation injury.
Pelvic irradiation has been a popular therapy modality for cervical cancer for many years, and its usage in rectal cancer and prostate cancer cases is on the rise. However, it is associated with significant side effects. In this study, we compared the different characteristics of surgical and nonsurgical patients who were treated for radiation enterocolitis, the treatment results, posttreatment quality of life (QOL), nutrition status, and predisposing factors for surgery.
From 1985 to 2009, the records of a total of 89 patients with chronic radiation enterocolitis in our hospital were retrospectively reviewed for demographic data, operative data and long-term treatment results. Posttreatment QOL and nutrition status were also recorded. Univariate and multivariate analyses were performed to identify the independent predicting factors associated with surgical intervention. Characteristics of surgical and nonsurgical patients were compared.
Radiotherapy before 1995, concomitant radiation uropathy and smoking were independent predictive factors for surgery. Surgical and nonsurgical cases had similar Kaplan–Meier curves. Although the recurrence rate of radiation enterocolitis was much higher for the surgical group (p = 0.031), both groups had similar QOL score (median: 8 vs.7; p = 0.709), serum albumin level (3.29 g/dL vs. 3.16 g/dL; p = 0.095), and body mass index (20.19 vs. 19.86; p = 0.603).
We confirmed that as compared with recently developed innovative techniques, early primitive radiotherapy techniques were associated with more severe radiotherapy complications that required surgery. Smoking may enhance patients’ vulnerability to severe radiation injury. Surgery for radiation-induced intestinal obstruction, intestinal fistula and perforation is warranted because QOL, serum albumin level and body mass index were similar between the surgical and nonsurgical groups.
Mast cells are an essential component of human radiation proctitis and contribute to experimental colorectal damage in mice
2011, American Journal of Pathology
Citation Excerpt :
Animals were anesthetized and a single 27-Gy (1.4 Gy/min) dose of gamma irradiation was delivered by a cobalt 60 source through a 1 × 0.8-cm window centered on the colorectal region. This model of localized single-dose radiation exposure does not directly simulate fractionation treatment, but generates histopathological lesions similar to those seen clinically (ie, severe acute mucosal ulceration and transmural collagen deposition during the late phase, with 100% rectal obstruction), as shown by Skwarchuk and Travis.21,22 At 2 and 14 weeks after exposure, the colon and rectum were fixed in 4% formaldehyde and embedded in paraffin.
Radiation proctitis is characterized by mucosal inflammation followed by adverse chronic tissue remodeling and is associated with substantial morbidity and mortality. Mast cell hyperplasia has been associated with diseases characterized by pathological tissue remodeling and fibrosis. Rectal tissue from patients treated with radiotherapy shows mast cell hyperplasia and activation, suggesting that these cells play a role in the development of radiation-induced sequelae. To investigate the role of mast cells in radiation damage, experimental radiation proctitis was induced in a mast cell-deficient (W^sh/W^sh) mouse model. The colon and rectum of W^sh/W^sh and wild-type mice were exposed to 27-Gy single-dose irradiation and studied after 2 and 14 weeks. Irradiated rodent rectum showed mast cell hyperplasia. W^sh/W^sh mice developed less acute and chronic rectal radiation damage than their control littermates. Tissue protection was associated with increased tissue neutrophil influx and expression of several inflammatory mediators immediately after radiation exposure. It was further demonstrated that mast cell chymase, tryptase, and histamine could change human muscularis propria smooth muscle cells into a migrating/proliferating and proinflammatory phenotype. These data show that mast cells have deleterious effects on both acute and chronic radiation proctitis, possibly by limiting acute tissue neutrophil influx and by favoring phenotypic orientation of smooth muscle cells, thus making them active participants in the radiation-induced inflammatory process and dystrophy of the rectal wall.
Radiation Injury
2010, Sleisenger and Fordtran’s Gastrointestinal and Liver Disease- 2 Volume Set: Pathophysiology, Diagnosis, Management, Expert Consult Premium Edition - Enhanced Online Features and Print
A Murine Model for the Study of Molecular Pathogenesis of Radiation Proctitis
2010, International Journal of Radiation Oncology Biology Physics
Citation Excerpt :
Partial bowel radiation in mice has been achieved with external beam radiation using tight collimation of the beam and large single fractions. However radiation effects were noted in the small bowel and the urethra (21, 22). These models are dissimilar to radiation treatment of pelvic malignancy in humans and, as a consequence, may have limited clinical relevance.
To establish a novel mouse brachytherapy model with which to study the role of inflammation in the pathogenesis of radiation proctitis.
The distal rectums of BALB/c and C57BL/6 mice were irradiated with three to five fractions of 5.5 to 8 Gy. Tissues were harvested and evaluated for histopathology, using the radiation injury score (RIS). Cytokine mRNA expression was assessed using real-time PCR.
Fifty percent of the mice treated with 22 Gy delivered in four fractions of 5.5 Gy died as a result of anorectal stenosis and distal bowel obstruction prior to the time of scheduled sacrifice, with a latency period of 4 to 10 weeks for the BALB/c and 3 to 4 weeks for the C57BL/6 mice. The RISs were 7, 12, and 8 at 2, 6, and 11 weeks, respectively, in the BALB/c mice and was 8.7 in the C57BL/6 mice on week 6. A 100- to 300-fold increase in interleukin-1β (IL-1β) (p = 0.04) and IL-6 mRNA (p = 0.07) and a 5- to 6-fold increase in transforming growth factor (TGF) and tumor necrosis factor-α mRNA expression levels (p < 0.001 and p = 0.01) were observed at 2 to 6 weeks after radiation. Cytokine mRNA tissue expression correlated positively with radiation dose (p < 0.0001). The RIS correlated well with IL-1β and IL-6 mRNA levels in the BALB/c mice and with IL-1β, IL-6, and TGF mRNA levels in C57BL/6 mice. Analysis of receiver operating characteristic curve showed that IL-1β and IL-6 have the largest area under the curve and therefore are good markers of radiation proctitis (p < 0.001).
Radiation-induced proctitis was associated with a dose-dependent, characteristic proinflammatory cytokine response pattern in a novel mouse model suitable for interventional studies.

View all citing articles on Scopus

^☆: Funding provided by NCI 06294 and the Rosalie B. Hite Predoctoral Scholarship Fund.

View full text

Biology ContributionsChanges in histology and fibrogenic cytokines in irradiated colorectum of two murine strains☆

Abstract

Introduction

Section snippets

Animals

Irradiation

Histology

Discussion

Acknowledgements

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Radiother Oncol

J Biol Chem

Gastroenterol

J Biol Chem

Cell

Gastroenterol

Bailliere’s Clin Gastroenterol

J Biol Chem

Int J Biochem Cell Biol

Radiother Oncol

Radiother Oncol

Int J Radiat Oncol Biol Phys

Int Rev Exp Pathol

Int J Radiat Oncol Biol Phys

Anal Biochem

Pathology

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Transforming growth factor betaA promotor of late connective tissue injury following radiotherapy?

Br J Radiol

Immunohistochemical localization of transforming growth factor beta and tumor necrosis factor alpha in the lungs of fibrosis-prone and “non-fibrosing” mice during the latent period and early phase after irradiation

Radiat Res

Radiation-induced alteration of rat mesangial cell transforming growth factor-β and expression of the genes associated with the extracellular matrix

Radiat Res

Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis

Radiat Res

Transforming growth factor beta in tissue fibrosis

N Engl J Med

Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor

EMBO J

Regulation of intestinal epithelial cell growth by transforming growth factor type beta

Proc Natl Acad Sci USA

Neutralisation of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring

J Cell Sci

Biology Contributions
Changes in histology and fibrogenic cytokines in irradiated colorectum of two murine strains☆