Departments of 1 Anatomical and Biomedical Sciences and 2 Physiology, University of Siena, 53100 Siena, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because the precise immunopathological events occurring in appendicitis are not completely understood, possible local production of endothelin-1 (ET-1) in human appendix was investigated. We used immunohistochemistry and in situ hybridization to detect the presence, distribution, and phenotype of ET-1-positive cells and prepro-ET-1 (pp-ET-1) mRNA-expressing cells. ET-1-positive stromal cells and pp-ET-1 mRNA-expressing cells were detected with different distributions and relative frequencies in normal control appendix, histologically normal appendix, and inflamed appendix. Six of 20 histologically normal appendixes from patients with a clinical diagnosis of acute appendicitis had many ET-1-positive stromal cells and high pp-ET-1 mRNA expression, similar to inflamed appendix. Forty percent of the pp-ET-1 mRNA-expressing cells were neutrophils, and the other positive cells were mast cells and macrophages. We suggest that local production of ET-1 by neutrophils and other inflammatory cells could be a molecular sign of focal inflammation in histologically normal appendixes and that ET-1 could be implicated, with other cytokines, in the pathogenesis of appendicitis by inducing appendiceal ischemia through vasoconstriction.
appendicitis; in situ hybridization; immunohistochemistry; neutrophils; mast cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ENDOTHELIN-1 (ET-1) is an endothelium-derived vasoconstrictor (9, 32) and consists of 21 amino acid residues with two intramolecular disulfide bonds. Ulcerative colitis and Crohn's disease are the two major disease entities of chronic relapsing inflammatory bowel disease. High ET-1 immunoreactivity has been shown in lamina propria and submucosa of patients with Crohn's disease and ulcerative colitis (19), and it has been suggested that local endothelin production by inflammatory cells may contribute to vasculitis in chronic inflammatory bowel disease by inducing intestinal ischemia through vasoconstriction (19). ET-1 also induces impairment of mucosal microvascular perfusion through activation of ETA receptors, causing significant tissue injury in the rat small intestine (17). The precise immunopathological events occurring in appendicitis are not completely known, and many appendixes removed for suspected appendicitis are subsequently classified as normal by conventional histological staining (30). There is an uncommon, enigmatic chronic appendicitis that shares histological features with typical Crohn's disease, but it presents as appendiceal disease. This unusual Crohn's diseaselike appendicitis has been called "Crohn's disease of the appendix" or "granulomatous appendicitis" (5, 10).
Local ET production by inflammatory cells may therefore be implicated in pathogenesis of appendicitis. In this study, we investigated the presence, distribution, and phenotype of ET-1+ cells and prepro-ET-1 (pp-ET-1) mRNA-expressing cells in human appendixes, using immunohistochemistry (IHC) and a nonradioactive in situ hybridization (ISH) method, respectively.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and samples. Thirty-three appendixes removed surgically from patients with a clinical diagnosis of acute appendicitis were studied. Twenty-six were subsequently classified as histologically normal, and seven were diagnosed as acute appendicitis. All studied subjects, according to their clinical history, were not suffering of peritonitis, hypertension, pulmonary hypertension, cirrhosis, renal failure, acute myocardial infarction, peptic ulcer, diabetes mellitus, or atherosclerosis. Three normal appendixes, removed from patients with colon cancer, served as control. Appendixes were divided into at least three segments from tip to base, fixed in Bouin's or formalin solution, and embedded in paraffin. Tissue sections (6-7 µm) were deparaffinized and washed with 0.1 M PBS (pH 7.4). ECV304, a human endothelial cell line, was used as a positive control. For ISH, the cells were harvested, washed in 0.1 M PBS, fixed in 4% paraformaldehyde for 30 min, washed in PBS, mixed with molten agar (2.5% in distilled water), and allowed to solidify. The cell bag was then routinely processed to produce a cell block from which paraffin sections were cut. For IHC, the cells, grown on chamber slides (Nalge Nunc, Naperville, IL), were fixed in 4% paraformaldehyde/5% acetic acid and permeabilized with 0.2% Triton X-100. Histological examination of hematoxylin and eosin stained appendix sections was carried out by the same histopathologist.
Probe preparation. The ET-1 probe was prepared according to the procedure described by Klein et al. (13), with minor modifications. Total RNA was extracted from 6 × 106 ECV304 (27), using "RNAeasy mini kit" (Quiagen, Hilden, Germany). Its integrity was checked on 1.6% (wt/vol) agarose gel, and the concentration was determined spectrophotometrically. To obtain cDNA, 5 µg of the extracted RNA were used as a template in the RT reaction, which was performed at 37°C for 30 min in a final volume of 25 µl and the ET-1 antisense specific primer 5'-GCT CTC TGG AGG GCT TGC-3'. Then, the cDNA of ET-1 was amplified in a PCR, using 200 ng of the sense primer 5'-CAG TTT GAA CGG GAG GTT TTT-3' and of the antisense primer used for the RT reaction. Amplification was performed in a thermal cycler (PCR Sprint, Hybaid, UK) under the following conditions: 94°C for 5 min, then 30 cycles at 93°C for 30 s, 56°C for 45 s, 72°C for 1 min, and finally 72°C for 5 min. The 645-bp ET-1 amplicon obtained was then labeled with digoxigenin (Dig) in a repeated PCR with the same ET-1 primers under the same conditions as before, apart from the addition of 70 µM Dig-labeled dUTP (Dig-11-dUTP, Boehringer Mannheim, Mannheim, Germany).
ISH. The appendix sections were dewaxed, rehydrated, and fixed in 4% paraformaldehyde in PBS pH 7.4 for 20 min and processed for hybridization as previously described (13). The sections were subsequently incubated with Fab fragments from sheep anti-Dig-alkaline phosphatase (APase) antibodies (150 U/200 µl; Boehringer) diluted 1:100 in PBS-BSA for 1 h at 37°C. APase color development was performed with Red (Vector), and endogen APase was inhibited by the addition of levamisole (Vector) in the substrate solution. Instead, the ECV304 slides were digested with 0.1% pepsin (Sigma, St. Louis, MO) in 0.01 M HCl at 37°C for 10 min. Hybrids were detected by application of, in sequence, mouse anti-Dig antibodies (2 µg/ml; Boehringer), peroxidase (PO)-conjugated rabbit anti-mouse immunoglobulins (13 µg/ml; DAKO, Milan, Italy), and PO-conjugated swine anti-rabbit immunoglobulins (26 µg/ml; DAKO). PO activity was revealed by development in 3-amino-9-ethylcarbazole (AEC; DAKO).
IHC.
The EnVision+ peroxidase detection system was used in an
immunoenzymatic technique (EnVision+ labeled polymer,
DAKO). The sections were incubated with 6%
H2O2 in H2O for 5 min followed by
3% PBS-BSA and then incubated overnight with appropriate primary
monoclonal antibodies (at working dilutions listed in Table
1). Immunoreactivity was visualized with
AEC or 3,3'-diaminobenzidine (DAB; DAKO).
|
Combined ISH and IHC. To define the immunophenotype of pp-ET-1 mRNA-expressing cells, we first used ISH combined with IHC in the same section of appendix, using APase and PO as reporter enzymes. Because all of the primary antibodies listed in Table 1 reacted with a cytoplasmic antigen, it was difficult to detect the ET-1 mRNA signal and the antigen specific for immunophenotyping simultaneously in the same cell, even using contrasting colors. After ISH procedure, pp-ET-1 mRNA-expressing cells were counted and photographed, and then the chromogen was removed with acetone without dehydration. For subsequent IHC, lactoferrin and CD68 antigen were detected by goat anti-mouse immunoglobulins (EnVision+ peroxidase). The tryptase antigen was detected with mouse anti-human antibody conjugated with APse (Chemicon, Temecula, CA). In this case, APase activity still present after the APase-Red reaction for ISH was inactivated by incubating the slides in 0.01 N HCl for 10 min at room temperature. The photographs obtained were printed on transparent plastic sheets, and the area with pp-ET-1 mRNA+ cells was superimposed on the areas immunostained for polymorphonuclear neutrophils (PMNs), mast cells (MCs), and macrophages (MØs).
ISH and IHC controls. Four negative control procedures were performed to assess the specificity of the ISH signal. 1) Before ISH, tissue sections were treated overnight with 300 µg/ml ribonuclease A (Boehringer) and 300 IU ribonuclease T1 (Boehringer) in Tris · HCl at 37°C. Tissue sections were treated; 2) omitting Dig-labeled probe; 3) omitting anti-Dig-APase antibodies; and 4) omitting Dig-labeled probe and anti-Dig-APase antibodies. After ISH, controls for IHC (in the same section) included omission of primary antibody and/or secondary antibody. In addition, ECV304, a human endothelial cell line, was used as a positive control.
Quantification of stained cells. The entire cross section of appendix was examined to evaluate the localization and relative frequencies of stained cells in the different compartments of the organ. All positive stained cells were counted (objective ×40) by two investigators who were unaware of the clinical groups. Results were presented as the number of stained cells per section, which were estimated semiquantitatively (+ = 1-5 cells, ++ = 6-20 cells, +++ = 21-50 cells, ++++ = 51-100 cells, +++++ = >100). The average tissue area of sections, measured by a computerized image analyzer (National Institutes of Health Image), was 2.5 × 107 ± 8% µm2.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemical localization of
ET-1+ cells in appendix.
Figure 1D' shows the positive
control, a human endothelial cell line, for immunocytochemical
detection of ET-1. Blood and lymphatic endothelial cells, epithelial
cells of the crypts and lining of the appendix lumen, lymphoid
follicles, muscularis mucosae and the two muscle layers, and myenteric
and submucous plexuses did not immunostain. On the contrary, stromal
cells with immunocytochemical staining of mature ET-1 in the cytoplasm
were detected (Fig. 1), but their distributions and relative
frequencies differed in the three clinical groups (Table
2).
|
|
Localization of pp-ET-1 mRNA-expressing cells in appendix by ISH.
To determine whether these ET-1+ stromal cells
produced ET-1 or took it up from the surrounding tissue, we used ISH to
detect pp-ET-1 mRNA-expressing cells in the same three groups
of appendixes. The intrinsic labeling of pp-ET-1-specific PCR product
with Dig-11-dUTP was evaluated by agarose electrophoresis (Fig.
2). The distributions and relative
frequencies of pp-ET-1 mRNA-expressing cells were similar to those of
mature peptide (Fig. 3 and Table 2).
Figure 3F shows the positive control for ISH.
|
|
Phenotypic characterization of pp-ET-1 mRNA-expressing cells.
Because PMN infiltration and increased mononuclear cell numbers are a
feature of acute appendicitis, and because MØs (6), enteric MCs (15), and neurons (11) are
reported to express ET-1, we evaluated the distribution of these cell
types using the monoclonal antibodies listed in Table 1. Figure
4 shows the mucosal distribution (between
the base of the crypts and the muscularis mucosae) of PMNs (Fig.
4A), MØs (Fig. 4B), and MCs (Fig. 4C)
in serial sections of the same specimen from one of the subset of six
histologically normal appendixes. Moreover, this subset of appendixes
showed more PMNs, MØs, and MCs compared with 20 histologically normal
appendixes, with similar distribution to ET-1+ cells
reported in Table 2.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have shown the presence of ET-1-positive stromal cells and pp-ET-1 mRNA-expressing cells and demonstrated their different distributions and relative frequencies in normal control appendix, histologically normal appendix, and inflamed appendix. In particular, a subgroup of histologically normal appendixes from patients with a clinical diagnosis of acute appendicitis had an increased number of ET-1-positive stromal cells and elevated pp-ET-1 mRNA expression, similar to the inflamed appendix. However, in this subgroup of histologically normal appendixes, there were more immunoreactive cells in the lamina propria than in the submucosa and muscularis, whereas the converse was true of inflamed appendix (more immunoreactive cells in the muscularis and submucosa than in the lamina propria). The lower incidence of positive cells in the mucosa of inflamed appendixes may be attributed to partial or complete destruction of the lamina propria by the inflammatory process.
Furthermore, our findings show clearly that the stromal pp-ET-1 mRNA-expressing cells are PMNs (40%) and MCs (25%). The ability of MCs and MØs but not of PMNs to synthesize and secrete ET-1 is well known (6, 15). Neutrophils are currently only regarded as being involved in the cleavage of exogenous big ET-1 to the biologically active peptide ET-1 (12, 26) and in the degradation of endothelin through release of proteases (12, 24). Identification of appendix PMNs as ET-1-producing cells is in line with our recent results that human activated PMNs, isolated from venous blood of normal donors, expressed pp-ET-1 mRNA and secreted appreciable levels of the mature ET-1 in the presence of lipopolysaccharide (2).
ET-1 immunoreactivity was not detected in the endothelium of blood and lymphatic vessels using two different primary monoclonal antibodies. This result is coherent with previous data in human colon (11) and probably reflects the tissue-processing method used. In fact, the human endothelial cell line, grown on chamber slides and fixed in paraformaldehyde/acetic acid, showed immunopositivity for ET-1 (Fig. 1D'). Besides, the same cells, fixed in paraformaldehyde, mixed with molten agar, and then embedded in paraffin express pp-ET-1 mRNA (Fig. 3F). Nevertheless, our processing method with Bouin's solution was optimal for demonstration of ET-1 immunoreactivity in inflammatory cells of the appendix. However, we cannot entirely exclude genetic predisposition and influences of appendix microenvironment. Indeed, regional differences in vascular endothelium structure, function, antigenicity, receptors, mRNA, and protein expression have been described (1, 23).
The results of the present study raise the question of the meaning of local ET-1 production by inflammatory cells and its possible role in the pathogenesis of appendicitis. Adeguate microvascular blood flow in the mucosa is considered essential to maintain mucosal integrity, and this blood flow is reduced during sepsis (7, 29) or hemorrage, leading to tissue damage. Elevated plasma levels of ET-1 in clinical septic conditions (25) suggest that ET-1 may play a role in the pathogenesis of various microvascular disorders (31) and intestinal mucosal injury. In fact, local intra-arterial infusion of low doses of ET-1 induces extensive ulceration and hemorrhagic damage of the gastrointestinal mucosa (18). Furthermore, increased ET-1 production and its receptor, ETA, are involved in the pathogenesis of endotoxin-induced intestinal mucosal damage (18) and high ET-1 immunoreactivity has been shown in lamina propria and submucosa of patients with Crohn's disease and ulcerative colitis (19).
During tissue ischemia and reperfusion (I/R), granulocyte
infiltration and mucosal dysfunction are coexisting phenomena, and the
role of neutrophils in microvascular injury has been described (8). Circulating PMN depletion and prevention of PMN
adherence significantly attenuate I/R-induced microvascular injury
(8), and depletion of extravascular mucosal PMN greatly
attenuates mucosal dysfunction (14). Thus these findings
suggest that PMNs mediate cell injury, exacerbating ischemic
damage by plugging the microvasculature and releasing undefined
cytotoxic substances (8). One of these mediators could be
ET-1, and several lines of evidence are consistent with this
hypothesis. First, activation of PMNs plays a central role not only in
response to invading pathogens, but also as an essential component of
the reaction to various forms of organ injury (3). PMNs
are activated by LPS through CD14, a
glycosylphosphatidylinositol-anchored protein (16), and
LPS-activated PMNs produce ET-1 (2). IFN- and TNF-
increase CD14 expression on PMNs and enhance LPS binding to PMNs
(28). Second, a subgroup of histologically normal
appendixes from patients with a clinical diagnosis of acute
appendicitis have abnormal TNF-
(30) and IFN-
(20) expression. Although PMNs are regarded as terminally
differentiated short-lived cells, they release cytokines such TNF-
(4) and IFN-
(33). Third, a recent study
suggests that infusion of ET-1 into the superior mesenteric artery
causes dose-dependent infiltration of PMNs, mucosal dysfunction in rat
small intestine, and neutropenia attenuates ET-1-induced increase in
mucosal permeability (21). Finally, ET-receptor blockers
reduce significantly I/R-induced intestinal mucosal injury and PMN
infiltration (22), indicating that tissue PMNs are
important mediators of ET-1-induced intestinal damage.
In conclusion, our results demonstrate the presence of pp-ET-1 mRNA and
its mature peptide in PMNs, MCs, and rare MØs, with different
distributions and relative frequencies in normal control appendix,
histologically normal appendix, and inflamed appendix. In particular, a
subgroup of histologically normal appendixes from patients with a
clinical diagnosis of acute appendicitis had many ET-1-positive cells
and high pp-ET-1 mRNA expression, similar to inflamed appendix. This
last result is in line with reports on the existence of a subgroup of
histologically normal appendixes with abnormal cytokine expression
(20, 30). We therefore suggest that local production of
ET-1 by neutrophils and other inflammatory cells could be a molecular
sign of focal inflammation in microscopically normal appendixes and that ET-1 could be implicated with other cytokines, such as TNF-,
IL-2, and IFN-
, in the pathogenesis of appendicitis by inducing
appendiceal ischemia through vasoconstriction.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: G. Grasso, Dept. of Anatomical and Biomedical Sciences, Via Aldo Moro, 53100 Siena, Italy (E-mail: grasso{at}unisi.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00262.2002
Received 2 July 2002; accepted in final form 25 October 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aird, WC,
Edelberg JM,
Weiler-Guettler H,
Simmons WW,
Smith TW,
and
Rosenberg RD.
Vascular bed-specific expression of an endothelial cell is programmed by the tissue microenvironment.
J Cell Biol
138:
1117-1124,
1997
2.
Cambiaggi, C,
Mencarelli M,
Muscettola M,
and
Grasso G.
Gene expression of endothelin-1 (ET-1) and release of mature peptide by activated human neutrophils.
Cytokine
14:
230-233,
2001[ISI][Medline].
3.
Caramelo, C,
López Farré A,
Riesco A,
and
Casado S.
Role of endothelin-1 in the activation of polymorphonuclear leukocytes.
Kidney Int
52, Suppl61:
S56-S59,
1997[ISI].
4.
Cassatella, MA.
The production of cytokines by polymorphonuclear neutrophils.
Immunol Today
16:
21-26,
1995[ISI][Medline].
5.
Dudley, TH, Jr,
and
Dean PJ.
Idiopathic granulomatous appendicitis or Crohn's disease of the appendix revisited.
Hum Pathol
24:
595-601,
1993[ISI][Medline].
6.
Ehrenreich, H,
Anderson RW,
Fox CH,
Rieckmann P,
Hoffman GS,
Travis WD,
Coligan JE,
Kehrl JH,
and
Fauci AS.
Endothelins, peptides with potent vasoactive properties, are produced by human macrophages.
J Exp Med
172:
1741-1748,
1990[Abstract].
7.
Gurll, NJ.
Organ blood flow and metabolism in shock: Overview.
Prog Clin Biol Res
299:
3-8,
1989[Medline].
8.
Hernandez, LA,
Grisham MB,
Twohig B,
Arfors KE,
Harlan JM,
and
Granger DN.
Role of neutrophils in ischemia-reperfusion-induced microvascular injury.
Am J Physiol Heart Circ Physiol
253:
H699-H703,
1987
9.
Hickey, KA,
Rubanyi G,
Paul RJ,
and
Highsmith RF.
Characterization of a coronary vasoconstrictor produced by cultured endothelial cells.
Am J Physiol Cell Physiol
248:
C550-C556,
1985[Abstract].
10.
Huang, JC,
and
Appelman HD.
Another look at chronic appendicitis resembling Crohn's disease.
Mod Pathol
9:
975-981,
1996[ISI][Medline].
11.
Inagaki, H,
Bishop AE,
Escrig C,
Wharton J,
Allen-Mersh TG,
and
Polak JM.
Localization of endothelinlike immunoreactivity and endothelin binding sites in human colon.
Gastroenterology
101:
47-54,
1991[ISI][Medline].
12.
Kaw, S,
Hecker M,
and
Vane JR.
The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes.
Proc Natl Acad Sci USA
89:
6886-6890,
1992[Abstract].
13.
Klein, SC,
Van Wichen DF,
Golverdingen JG,
Jabobse KC,
Broekhuizen R,
and
de Weger RA.
An improved, sensitive, non-radioactive in situ hybridization method for the detection of cytokine mRNAs.
Acta Pathol Microbiol Immunol Scand
103:
345-353,
1995.
14.
Kubes, P,
Hunter J,
and
Granger DN.
Ischemia/reperfusion-induced feline intestinal dysfunction: importance of granulocyte recruitment.
Gastroenterology
103:
807-812,
1992[ISI][Medline].
15.
Liu, Y,
Yamada H,
and
Ochi J.
Immunocytochemical studies on endothelin in mast cells and macrophages in the rat gastrointestinal tract.
Histochem Cell Biol
109:
301-307,
1998[ISI][Medline].
16.
Luchi, M,
and
Munford RS.
Binding, internalization, and deacylation of bacterial lipopolysaccharides by human neutrophils.
J Immunol
151:
959-969,
1993
17.
Massberg, S,
Boros M,
Leiderer R,
Baranyi L,
Okada H,
and
Messmer K.
Endothelin (ET)-1 induced mucosal damage in the rat small intestine: role of ETA receptors.
Shock
9:
177-183,
1998[ISI][Medline].
18.
Miura, S,
Fukumura D,
Kurose I,
Higuchi H,
Kimura H,
Tsuzuki Y,
Shigematsu T,
Han JY,
Tsuchiya M,
and
Ishii H.
Roles of ET-1 in endotoxin-induced microcirculatory disturbance in rat small intestine.
Am J Physiol Gastrointest Liver Physiol
271:
G461-G469,
1996
19.
Murch, SH,
Braegger CP,
Sessa WC,
and
MacDonald TT.
High endothelin-1 immunoreactivity in Crohn's disease and ulcerative colitis.
Lancet
339:
381-385,
1992[ISI][Medline].
20.
Muscettola, M,
Cambiaggi C,
Mencarelli M,
Carbotti P,
Massai L,
Migliaccio P,
and
Grasso G.
IFN-, IFN-
receptor, IFN-
mRNA, and STAT1 mRNA in human appendix.
Eur Cytokine Netw
11:
192,
2000.
21.
Oktar, BK,
Coskun T,
Bozkurt A,
Yegen BÇ,
Yüksel M,
Haklar G,
Bilsel S,
Aksungar FB,
Çetinel S,
Granger DN,
and
Kurtel H.
Endothelin-1-induced PMN infiltration and mucosal dysfunction in the rat small intestine.
Am J Physiol Gastrointest Liver Physiol
279:
G483-G491,
2000
22.
Oktar, BK,
Gulpinar MA,
Bozkurt A,
Ghandour S,
Çetinel S,
Moini H,
Yegen BÇ,
Bilsel S,
Granger DN,
and
Kurtel H.
Endothelin receptor blockers reduce I/R-induced intestinal mucosal injury: role of blood flow.
Am J Physiol Gastrointest Liver Physiol
282:
G647-G655,
2002
23.
Page, C,
Rose M,
Yacoub M,
and
Pigott R.
Antigenic heterogeneity of vascular endothelium.
Am J Pathol
141:
673-683,
1991[Abstract].
24.
Patrignani, P,
Del Machio A,
Bazzoni G,
Daffonchio L,
Hernandez A,
Modica R,
Montesanti L,
Volpi D,
Patrono C,
and
Dejana E.
Inactivation of endothelin by polymorphonuclear leukocyte-dereived lytic enzymes.
Blood
78:
2715-2720,
1991[Abstract].
25.
Pittet, JF,
Morel DR,
Hemsen A,
Gunning K,
Lacroix JS,
Suter PM,
and
Lundberg JM.
Elevated plasma endothelin-1 concentrations are associated with the severity of illness in patients with sepsis.
Ann Surg
213:
231-264,
1991.
26.
Sessa, WC,
Kaw S,
Hecker M,
and
Vane JR.
The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes.
Biochem Biophys Res Commun
174:
613-618,
1991[ISI][Medline].
27.
Takahashi, K,
Sawasaki Y,
Hata JJ,
Mukai K,
and
Goto T.
Spontaneous transformation and immortalization of human endothelial cells.
In Vitro Cell Dev Biol
25:
265-274,
1990.
28.
Takeshita, S,
Nakatani K,
Takata Y,
Kawase H,
Sekine I,
and
Yoshioka S.
Interferon-gamma (IFN-) and tumour necrosis factor-alpha (TNF-
) enhance lipopolysaccharide binding to neutrophils via CD14.
Inflamm Res
47:
101-103,
1998[ISI][Medline].
29.
Wang, P,
Zhou M,
Rana MW,
Ba ZF,
and
Chaudry IH.
Differential alterations in microvascular perfusion in various organs during early and late sepsis.
Am J Physiol Gastrointest Liver Physiol
263:
G38-G43,
1992
30.
Wang, Y,
Reen DJ,
and
Puri P.
Is a histologically normal appendix following emergency appendicectomy always normal?
Lancet
347:
1076-1079,
1996[ISI][Medline].
31.
Wilson, MA,
Steeb GD,
and
Garrison RN.
Endothelins mediate intestinal hypoperfusion during bacteremia.
J Surg Res
55:
168-175,
1993[ISI][Medline].
32.
Yanagisawa, M,
Kurihara H,
Kimura S,
Tomobe Y,
Kobayashi M,
Mitsui Y,
Yazaki Y,
Goto K,
and
Masaki T.
A novel potent vasoconstrictor peptide produced by vascular endothelial cells.
Nature
332:
411-415,
1988[ISI][Medline].
33.
Yeaman, GR,
Collins JE,
Currie JK,
Guyre PM,
Wira CR,
and
Fanger MW.
IFN- is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils.
J Immunol
160:
5145-5153,
1998