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RAPID COMMUNICATION
A Bone Marrow Stromal-Derived Growth Factor, Interleukin-11, Stimulates
Recovery of Small Intestinal Mucosal Cells After Cytoablative Therapy
By X.X. Du, C.M. Doerschuk, A. Orazi, and D.A. Williams
The proliferation of epithelial cells lining
the small intestinal
mucosa may be regulated by microenvironmental signals
leading to differentiation of precursor cellsin the small intestinal crypts. Proliferation of hematopoietic cells within
the hematopoietic microenvironment is
known to be regulated by a growing numberof glycoprotein growth factors
in a hierarchial fashion.W e studied the effects of administration of the microenvironment-derived hematopoietic
growth factor interleukin-l 1 (IL-11) on mice given combination radiation/chemotherapy. Treatment ofsuch mice
with IL-l1 led to significantly increased survival and evidence of rapid recovery of
the small intestinal mucosa,
which is severely damaged by these cytoxic agents. This
recovery was associated with an increase in the mitotic index ofcrypt cells and
an increased frequency of staining of
these cells with a monoclonal antibodyto proliferating cell
nuclear antigen, a member of the cyclin family of nuclear
antigens.
0 1994 by The American Society of Hematology.
R
ECENT MOLECULAR cloningof a variety of hematopoietic growth factors and the increasing use of
some of these proteins in clinical medicine has led to decreased morbidity of individuals with both congenital and
acquired bone marrow (BM) hypoplastic conditions.’ Interleukin-l 1 (IL-l l), a growth factor isolated and cloned
from an immortalized primate BM stromal cell line,*.’ has
been shown to stimulate megakaryocyte, erythroid, and myeloid colonies and the number of immunoglobulin-secreting B lymphocytes in vitro2x4,’and in vivo6 as well as accelerate the recovery of murine peripheral blood neutrophils
and platelets, bone marrow cellularity, and myeloid/mixed
In addiprogenitors in vivo after myeloablative therapie~.”~
tion, IL-1 I has been shown to increase peripheral platelet
counts when administered to normal mice.” Nonhematologiceffectsof IL-11 includeinhibition of preadipocyte
differentiation in cell lines and in primaryhuman long-term
marrow
These activities are distinct from the
effects of several growth factors currently approved for use
in the clinical setting or undergoing clinical trials.
Individuals receiving cytoablative therapy for cancer and
leukemia as well as in preparation for BM transplantation
are at risk for developing serious infections and bleeding.I4
The infectious complications of such therapies arerelated to
prolonged and severe neutropenia, as well as damageto the
small intestinal mucosa bamer, leading to entry of gastrointestinal flora into the blood.” Previous studies have documented that acute injury to rodent small intestinal mucosa
from either chemotherapy or radiation leads to shortening
of small intestinal mucosa villus length.I6-l8 Thisacute
change is caused by cell death and continued migration of
epithelial cells toward the apex of the villi in the absence of
mitotic activity in the crypt base to replace differentiating
ceIls.’*J9
To assess the functional significance of IL- 1 1 administration in vivo, mice were given combination radiation/chemotherapy (combinedmodality, CM, therapy) with or without L 1 1 administration. These animals are highly
susceptible to sepsis from endogenous gastrointestinal bacteria caused by severe damage to the small intestinal mucosa. We found that IL- 1 1 treatment results in a significant
increase in survival of mice given combination radiation/
chemotherapy. This increase in survival is associated with
Blood, Vol83, No 1 (January l ) , 1994: pp 33-37
Fig 1. Survival of mice after combined chemotherapy/radiation.
Results are fromthree independent experimentswith a total of 13
mice in each group.
decreased bacterial foci in the liver, spleen, and mesentery,
increased small intestinal villus (SIV) length, increased villus/crypt ratio, and increased cycling of crypt cells. We conclude that IL- I 1 administration is associated with enhanced
From the HermanB. Wells Centerfor Pediatric Research, James
Whitcomb Riley HospitalforChildren; and the Section ofpediatric
Pulmonology, the Department of Pathology, Howard Hughes Medical Institute, Indiana University School of Medicine, Indianapolis.
Submitted October 7, 1993; accepted October8,1993.
Supported by National Institutes of Health Grants No. R01
HL46528 (D.A. W.) and P014645168 (X.X.D.). D.A. W. receives
payments from Children’s Hospital (Boston, MA) based on certain
milestones set forth in an IL-11 agreement between Genetics Institute (Cambridge, MA) and Children’s Hospital.
Address reprint requests to David A. Williams, MD, Howard
Hughes Medical Institute, Herman B Wells
Centerfor Pediatric Research, Indiana University School ofMedicine, 702 Barnhill Dr, Indianapolis, IN 46202-5225
The publication costs of this article were defayed in part by page
charge payment.This article must therefore be hereby marked
“advertisement” in accordance with18 U.S.C. section 1734 solelyto
indicate thisfact.
0 1994 by The American Society of Hematology.
0006-4971/94/8301-0037$3.00/0
33
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DUET AL
34
recovery of small intestine mucosa after otherwise lethal
doses of combined cytotoxic therapy. Therefore,
IL-11may
prove useful in accelerating the recovery of both BM and
small intestinal epithelial
cells after cancer therapy.
MATERIALSANDMETHODS
Mice and cytoablutive therapies. Eight-week-old to IO-weekold C3H/HeJ mice (Jackson Laboratory, Bar Harbor, ME) were
administered 5-fluorouracil (5-W)(diluted in Hanks' Balanced
Salt Solution [HBSS] containing 0.025 mol/L HEPES) at 150 mgl
kg body weight intraperitoneal (IP) 3 days beforesublethal irradiation (6.0 Gy total body irradiation delivered from a Siemens 250
KVp x-ray therapy machine (Madison, WI), filtered with 1.O mm
Cu, giving a half value layer of 2.1 mm Cu at 50 cm source skin
Fig 3. PCNAstaining of smallintestinalcrypts.(A)Vehiclatreatedcontrolmice. (B)11-11 treatedmice.Thefigureshows
2 days after irradiation.
PCNA staining from mice killed
Fig 2. Histologic sectionsof small intestine.(A) Vehicle-treated
control mice. (B) Mice treated with IL-11. The figure shows small
intestineof mice killed6 days after irradiation.
distance (SSD), and with a dose rate of 78.13 cGy/min). No BM
infusions were givento these animals. Recombinant human IL-l1
(rhIL-1 l ) was administered beginning on the same day as irradiation. rhIL-l1 (Genetics Institute, Cambridge, MA) was diluted in
HBSS (GIBCO, Grand Island, N Y ) containing 0.1% bovine serum
albumin (BSA) (wt/vol; Boehringer-Mannheim, Indianapolis, IN)
and 0.025 mol/L HEPES(GIBCO). RhIGl1 (250 &kg body
weight) was injected subcutaneously in 0.2-mL v01 twice per day
starting on the same d a y as irradiation. Control mice received the
same volume of HBSS/O. l % BSA (vehicle injections).In some ex(usperiments, mice were irradiated with 7.0,7.5, and 8.0 Gy y-ray
ing "7Cs source at 95.83 cGy/min) as above.
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INTESTINAL
IL-l1 STIMULATES
35
CELLS
Table 1. Effect ofIL-l1 on Small Intestine Crypt Cell Recovery Post5-FU and Irradiation
Villus Length
Crypt Depth
Day
1
14.3
123.4k2
3
141.5f11.9
4
120.6f 5
Crypt/Villus
BSA
IL- 1 1
BSA
IL-l 1
BSA
IL-l 1
76.8 f 9.2
71 . l f 4.7
129.0 f 7.8
141.8119.9
118.5 k 14.0
117.817.7
398.6 f 45.4
491.9 k 23.2
468.7 ? 47.4
500.2 f 51.3
523.2 ? 17.4
479.0 1 31.8
603.4 1 41.9t
533.9 f 24.2
635.0 f 26.9t
661 5k20.1'
0.20 ? 0.01
0.25 f 0.01
0.33 f 0.01
0.29 ? 0.00
0.23 f 0.00
0.15f0.00'
0.22 f 0.01t
0.27 f 0.01'
0.19tO.00'
0.18f0.01'
154.5 t 10.2
11.7
Each number represents data from 3 animals; 10 crypts/animal, 10 villi/animal
* P < .O1 versus BSA group.
t P < .05 versus BSAgroup.
Histologic and morphometric analysis. Mice dying or killed after combined radiation/chemotherapy were autopsied and tissues
fixedin 10% buffered formalin overnightwithin 12 hours aRer
death. Tissues from each organ (liver, spleen, kidney,intestine
small
and mesentery, abdominal wall, lung,heart, and femurs) wereembedded in paraffin wax usingstandard techniques. Four-micron sections were cut and stained with hematoxylin/eosin. Ten independent measurements of villus height,crypt depth, and metaphases/
crypt per specimen of small intestine were made from mice killed
daily using an objective mounted micrometer at 200 X magnification. Hepatic bacterial foci were counted both macroscopically as
surface coloniesand microscopicallyon randomly chosen liver histologic sections.
Zmmunohislochemistry. Proliferating
cell
nuclear
antigen
(PCNA) immunohistochemicalstainingwas performedon 2-cm jejunum sections obtained from CM mice killed daily from day 1
through day 5 postirradiation (day 4 through 8 post-5-W). Tissue
was fixedin 10%formalin for 6 to 12 hours, paraffin embedded, and
sections were dried on polylysine-treated glass slides. Slides were
then deparaffinized and rehydrated. Endogenous peroxidase was
quenched by a 5-minute incubation in 3% hydrogen peroxide.The
slides were covered withnormal goat serum for 20 minutes, incubated overnight at 4°C with PC10 antibody against PCNA (1530;
Dako, Santa Barbara, CA),and then stained for 30 minutes with a
biotin-conjugated goat-antimouse antibody, followed by peroxidase-conjugated streptavidin (both Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 30 minutes. The enzyme was developed with 3,Ydiaminobenzidine (Sigma, St Louis, MO). PCNA+
nuclei stained brown. The percentage ofPCNA' crypt cellsand absolute number of PCNA' cells percrypt were measured by counting
20 randomly chosen crypts/section/mouse.
Table 2. Effect of I L - l 1 on Proliferation of SmallIntestine Crypt
Cells asQuantitated by PCNA Staining
IL- 1 1
BSA
Day
1
2
2.4
3
4
5
% o f +Nuclei
+Nuclei/Crypt
17.0t6.8
3.1 k 1.4
0.4
k 0.3
1.9 k 1.3
1 1.7
0.8 k 0.5
0.4
k 0.2
4.2
1.3 t 0 . 4
1 . 7 f 0.4
0.6 k 0.1
10.5
%of +Nuclei
+Nuclei/Crypt
1.2
18.4t 9 . 25 . 0 f
10.5 k 1 .O'
2.6 0.3'
7 . 6 f 1.9t
2.1 f 0 . 5 t
k 1.5t
1.3
f 0.4t
k 11.3
3.3
f 3.4
Each number represents data from 3 animals; 20 crypts/animal, 400
to 600 nuclei.
P < .01 versus BSA group.
t P < .05 versus BSAgroup.
RESULTS
After treatment with 5-FU,'4 and sublethal doses of Xirradiation, damageto thesmall intestine of C3H/HeJ mice
is extensive and themajority of mice die within 10 days (Fig
l). Dying animals show wasting, diarrhea, and tilting and
rotating indicative of centralnervous system infection.
Treatment of mice with 250 pg/kg/d of rhIL11 was associated with significant increase in survival after thiscytoablative therapy, inspite of no increase in peripheral neutrophil
counts or BM myeloid progenitors (Fig I). Additional experimentsin which increasing doses and an alternative
source of irradiation (I3'Cs) were administered confirmed
the increase in survival in IL-1 l-treated mice over several
dose ranges. The experiments tested radiation doses of 6.0,
7.0,7.5, and 8.0 Gy (all after 150 mg/kg 5-FU) and included
a total of 2 15 mice. Survival in the control group in all experiments was 27% compared with 64% inthe IL-11-treated
mice (P< .0001). IL-l1 treatment was associated with a
markedreductionin
thenumber of bacterial foci in
multiple organs with none detectable in many mice after
sacrifice(data notshown). Microbiologic analysis of foci dissected from the liver of control mice uniformly showed that
the causative organism was Escherichia coli, a common enteric organism in mice.
To determine the potential source of E coli organisms
shown in mice after CMtreatment, histologic sections ofthe
small intestine were examined in controland IL-l l-treated
mice sacrificed daily after irradiation. The small intestinal
mucosa of control mice showed marked destructionof villus
structure with shortening of the villus length, vacuolization
and pyknotic nuclear structures (Fig 2A). In contrast, IL1 l-treated mice showed mild changes in morphology of the
SIV (Fig 2B). Morphometric analysis of crypt and villi
length showed a significant increase in the ratio of the crypt
depth/villi length in IL- I l-treated mice compared with
control mice (Table 1). Villus shortening was most prominent 24 hours after irradiation in both groups
of mice. Villus
length recovered quickly in IL-I l-treated mice, while remaining abnormal in surviving control mice through day 9
(data not shown). The changes shown in control mice after
CM treatment were qualitatively similar to changes noted
previously after treatment with either 5-FUl6or radiation'*
alone, althoughthe magnitude of the damage and thelength
of time torecovery in the few control animals thatsurvive
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36
DU ET AL
over the first week are larger and longer than previous studies. These differences may be caused by the combination
of high-dosechemotherapy followed by high-dose radiation
during thepostchemotherapy recovery phase.
Because the villus length is dependent on proliferation
and differentiation of crypt stem and progenitor cell^,'"'^
the mitotic index of crypt cells was determined in control
and IL-l l-treated mice killed daily after irradiation. Significant increases in the number of mitoses/crypt (2.0 f 0.5
v 0.9 2 0.4, IL- 1 I v control, P < .OO 1) as well as in the number of mitoses/100 pm of epithelial basement membrane
(0.22 f 0.06 v 0.10 f 0.04, P < .O 1) were seen on day 5 after
irradiation in the IL- 1 1-treated mice. Control mice surviving to day 9 postirradiation showed slightly increased numbers of mitotic crypt cells, but the numbers were still depressed compared with normal or 1L-l l-treated mice.
The increase in cell cycle activity after CM treatmentand
IL-l I administration was further characterized by staining
with PC-10,20-22amonoclonalantibody directed against
PCNA, a member of the cyclin family of nuclear proteins
(Table 2 and Fig 3). IL-I I administration was associated
with a twofold to fivefold increase in the number of crypt
cell nuclei staining with PC-IO on days 2 through 4 after
irradiation. Taken together, these data show that IL-l 1 administration to mice after severe damage to the small intestinal crypt cells hastens recovery of the villus structure because of increased proliferation of presumably the crypt
progenitor cell.
DISCUSSION
Cytotoxic agents used in BM transplantation and cancer
therapy affect rapidly proliferating cells in boththe BM and
small intestine, leading to severe and often dose-limiting
toxicities. IL- I 1 has been shown to have pleiotropic effects
on reconstitution after ablative therapy depending on the
model e ~ a m i n e d . ~The
- ' ~accelerated
~~~
recovery of peripheral blood counts noted inthe above studies are likely
caused by effects ofIL- 1 1 on hematopoietic progenitor compartments. Girasole et al have also shown induction of osteoclast formation and stimulation of bone resorption in
mice treated with IL-l1 (G. Girasole, personal communication, October, 1993).
IL-l1 has also been reported to have effects on several
nonhematopoietic tissues. Kawashima et all3 cloned an
identical protein, adipogenesis inhibitory factor, based on
inhibition of fat accumulation in 3T3-Ll cells. Such inhibition of fat accumulation has also been shown in primary
human long-term marrow cultures and was associated in
these cultures with the apparent inhibitionof differentiation
of preadipocytes in the hematopoietic microenvironment.' '
Mehler et aIz4have shown that IL- 1 1 has effects on neuronal
differentiation. From these studies, it is clear that IL-l l has
effects on a wide variety of tissues, particularly mesenchymal cells.
Several growth factors, including IL- l 25 and Steel factor,26
have been shown to be radioprotective in murine studies.
Radioprotection by these growth factors has been reported
to be the result of changes in the progeny of BM stem and
progenitor cells. In contrast,in the study reported here,
analysis of small intestinal mucosa showed rapid recovery
of villi length and increased proliferative activity within the
crypt cells of IL-I l-treated mice compared with control
mice, although it remains unclear whether this is a direct
effect or one mediated through accessory cells. IL-11 may
be a unique protein that has regulatory roles in a variety of
complex environments containing stromal cells. The positive effects on the recovery of several tissues exhibiting doselimiting toxicities after cytoablative therapies may prove
clinically useful in the future.
ACKNOWLEDGMENT
We thank Drs Steven Clark, Sandy Goldman, and Gary Varilek
for helpful discussions.
REFERENCES
1. Clark SC, Kamen R: The human hematopoietic colony-stimdating factors. Science 236: 1229,1987
2. PaulSR, Bennett F, Calvetti JA, Kelleher K, Wood CR,
OHara RM Jr, Leary AC. Sibley B, Clark SC, Williams DA, Yang
Y-C: Molecular cloning of a cDNA encoding interleukinI l , a stromal cell-derived lymphopoietic and hematopoietic cytokine. Proc
Natl Acad Sci USA 87:75 12, 1990
3. Paul SR, Yang Y-C, Donahue RE, Goldring S, Williams DA:
Stromal cell-associated hematopoiesis: Immortalization and characterization of a primate bone marrow-derived stromal cell line.
Blood 77: 1723, I99I
4. Quesniaux VFJ, Clark SC, Turner K, Fagg B: Interleukin-l 1
stimulates multiple phases of erythropoietin in vitro. Blood 80:
1218, 1992
5. Schibler KR, Yang Y-C, Christensen RD: Effect of interleukin-l 1 on cycling status and clonogenic maturation of fetal and
adult hematopoietic protenitors. Blood 80:900, I992
6. Yin T, Schendel P, Yu-Chung Y: Enhancement of in vitroand
in vivo antigen-specific antibody responses by interleukin- I 1 . J Exp
Med 175:21 I , 1992
7. Du XX, Neben T, Goldman S, Williams DA: Effects of recombinant human interleukin-l 1 on hematopoietic reconstitution
in transplant mice: Acceleration of recovery of peripheral blood
neutrophils and platelets. Blood 8 1:27, 1993
X. Du XX, Keller DC, Maze R, Williams DA: Comparative
effects of in vivo treatment using interleukin- 1 I and stem cell factor
on reconstitution in mice following bone marrow transplantation.
Blood82:1016, 1993
9. Hangoc G, Yin T, Cooper S, Schendel P, Yang Y-C, Broxmeyer HE: In vivo effectsof recombinant interleukin-l 1 on myelopoiesis in mice. Blood 8 1 :965, 1993
IO. Neben TY, Loebelenz J, Hayes L, McCarthy K, Stoudemire
J, Schaub R, Goldman SJ: Recombinant human interleukin-l 1
stimulates megakaryocytopoesis and increases peripheral platelets
in normal and splenectomized mice. Blood 8 1:901, 1993
I 1. Keller DC, Du XX, Srour EF, Hoffman R, Williams DA:
Interleukin- I I inhibits adipogenesis and stimulates myelopoiesis in
long-term marrow cultures. Blood 82: 1428, 1993
12. Yin T, Miyazawa K. Yang YC: Characterizationof interleukin-l 1 receptor and protein tyrosine phosphorylation induced by interleukin-l I in mouse 3T3-LI cells. J Bio Chem 267:
8347. 1992
13. Kawashima I, Ohsumi J, Mita-Honjo K, Shimoda-Takano
K, Ishikawa H, Sakakibara S, Miyadai K, Takiguchi Y : Molecular
cloning of cDNA encodingadipogenesis inhibitory factor and identity with interleukin- I 1. FEBS Lett 283: 199, 199 1
14. Siber GR, Mayer RJ. Levin MJ: Increased gastrointestinal
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IL-11 STIMULATESINTESTINALMUCOSAL
CELLS
absorption of large molecules in patients after 5-fluorouracil therapy for metastatic colon carcinoma. Cancer Res 40:3430, 1980
15.Berg R D Bacterial translocation from the gastrointestinal
tract of mice receiving immunosuppressive chemotherapeutic
agents. Curr Microbiol8:285, 1983
16. Ijiri K, Potten CS: Response of intestinal cells of differing
topographical and hierarchical status to ten cytotoxic drugs and five
sources of radiation. Br J Cancer 47: 175,1983
17. AI-Dewachi HS, Wright NA, Appleton DR, Watson AJ: The
effect of a single injection of hydroxyureaon cell population kinetics
in the small bowel mucosaofthe rat. Cell Tissue Kinet10203, 1977
18. Potten CS: The role of stem cells inthe regeneration of intestinal crypts after cytotoxic exposure, in Buttenvorth BE, Slaga TJ,
Farland W, McClain M (eds): Chemically induced cell proliferation
implications for risk assessment, New York, NY, Wiley-Liss, 1989,
p 155
19. Chwalinski S, Potten CS: Crypt base columnar cells in ileum
of BDF, male mice-Their numbers andsome features oftheir proliferation. Am J Anat 186:397, 1989
20. Hall PA, Levison DA, Woods AL, Yu CC, Kellock DB, Watkins JA, Barnes DM, Gillett CE, Camplejohn R, Dover R, Waseem
37
NH, Lane D P Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with
evidence of deregulated expression in some neoplasms. J Pathol
162:285, 1990
2 1. Zeymer U, Fishbein MC, Forrester JS, Cercek B: Proliferating cell nuclear antigen immunohistochemistry in rat aorta after
balloon denudation. Am J Pathol 141:685, 1992
22. Garcia RL, Coltrera MD, Gown AM: Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed,
embedded tissues. Am J Pathol 134:733, 1989
23. Anderson KC, Morimoto C, Paul SR, Chauhan D, Williams
D, Cochran M, Barut BA: Interleukin-l I promotes accessory celldependent B-cell differentiation in humans. Blood 80:2797, 1992
24. Mehler MF, Rozental R, Dougherty M, Spray DC, Kessler
JA: Cytokine regulation ofneuronal differentiation of hippocampal
progenitor cells. Nature 362:62, 1993
25. Neta R, Douches S, Oppenheim JJ: Interleukin-l is a radioprotector. J Immunol 136:2483, 1986
26. Zsebo KM, Smith KA, Hartley CA, Greenblatt M, Cooke K,
Rich W, McNieceIK: Radioprotection of mice by recombinant rat
stem cell factor. Proc Natl Acad Sci USA 89:9464, 1992
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1994 83: 33-37
A bone marrow stromal-derived growth factor, interleukin-11,
stimulates recovery of small intestinal mucosal cells after
cytoablative therapy
XX Du, CM Doerschuk, A Orazi and DA Williams
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