Department of Obstetrics and Gynecology, Ehime University School of Medicine, Shitsukawa, Shigenobu, Ehime 791-0295, Japan
1 To whom correspondence should be addressed. e-mail: tkatayam{at}m.ehime-u.ac.jp
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Abstract |
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Key words: endothelial permeability/intracellular adhesion molecule (ICAM)-1/leukocyte behaviour/microcirculation/ovarian hyperstimulation syndrome (OHSS)
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Introduction |
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Certain inflammatory cytokines (interleukin-6, tumour necrosis factor- and interleukin-1) and growth factors such as vascular endothelial growth factor may play major roles in the pathophysiology of systemic acute-phase responses (Sirois and Edelman, 1997
; Tamion et al., 1997
). On the other hand, a rapidly expanding body of data has highlighted the importance of endothelial cell adhesion molecules in development and propagation of inflammatory processes in many human tissues (Simmons et al., 1988
; Springer et al., 1990
; Bevilacqua et al., 1993
; Carlos et al., 1994
). These molecules facilitate leukocyte adhesion and extravasation through the vessel wall, which are key steps in response to infection and tissue injury. Adhesion molecules such as vascular cell adhesion molecule-1 and intracellular adhesion molecule (ICAM)-1, are transmembrane glycoproteins that are members of the immunoglobulin superfamily (Aoki et al., 1997
; Iigo et al., 1997
; Morisaki et al., 1997
). This group of molecules includes major mediators of white blood cell adhesion, interaction and extravasation during inflammatory and immune reactions. Recent reports indicate that amounts of ICAM-1 in the blood correlate with biological and clinical aspects of severe OHSS (Daniel et al., 1999
; Abramov et al., 2001
; Bonello et al., 2002
). OHSS may be seen as a disorder where adhesion molecules are involved in exaggerated leukocyte recruitment and transendothelial migration, causing tissue damage and capillary hyperpermeability.
Although occurrence of increased vascular permeability is not limited to ovulation, OHSS can be viewed as an exaggeration of events that occur during the menstrual cycle (Cavender et al., 1988). Investigation of changes in microvascular permeability at ovulation, therefore, is important to understanding the cause of OHSS. Until now, little has been known about sequential multistep leukocyteendothelium interactions and albumin leakage from endothelial cells in the microcirculation at induction of ovulation.
We therefore used intravital microscopy to examine the microvascular permeability and behaviour of leukocytes involving adhesion molecules in the rat mesenteric microcirculation accompanying induction of ovulation (Suematsu et al., 1995; Katayama et al., 2000
).
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Materials and methods |
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Intravital observation of neutrophil adhesion and FITC-labelled albumin leakage in rat mesenteric venules
After rats were anaesthetized with pentobarbital sodium (40 mg/kg, i.m.), the femoral vein was cannulated with a polyethylene catheter (Atom, Tokyo, Japan) for infusion of monoclonal antibody (mAb) and fluorescein isothiocyanate-labelled bovine serum albumin (FITC BSA). The abdomen was opened via a midline incision, and the ileocaecal portion of the mesentery was gently exposed and mounted on a plastic stage. Postcapillary venules 2040 µm in diameter were selected for evaluation of leukocyte adhesion as described previously (Suematsu et al., 1989), and were visualized with an intravital microscope (BX50: Olympus, Tokyo, Japan) assisted by a high-resolution colour charge-coupled device (CCD) camera (HCC-600: FLOVEL, Tokyo, Japan). The mesentery was superfused continuously with KrebsHenseleit bicarbonate-buffered solution saturated with 95% N2/5% CO2 at 37°C. Centreline erythrocyte velocity (VR) in selected venules was monitored continuously by a temporal correlation velocimeter (Instrumentation for Physiology and Medicine, San Diego, CA). To minimize effects of surgical insults on observed leukocyte adhesion, the preparation was allowed to stabilize for 30 min after abdominal surgery. Leukocyte behaviour in venules was videotaped continuously by an S-VHS video recorder (RS-232C: Panasonic, Tokyo, Japan). The density of adherent leukocytes was expressed as their number per 100 µm segment of venule. Adherent leukocytes were defined as those remaining stationary for >30 s in the same portion of the selected venular segment. Rolling leukocytes were defined as those moving at a velocity less than that of erythrocytes in the same vessel. White blood cell velocity, VW, was determined as the time required for a leukocyte to transverse a given length of venule, as described previously (Morisaki et al., 2001
, 2002). Venular wall shear rates (
) were calculated according to the formula
= 5(VR/DV), with DV representing venular diameter, as described elsewhere (House et al., 1987
).
Vascular albumin leakage was quantified using a previously reported protocol (Kubes et al., 1996). Briefly, FITCBSA (25 mg/kg: Sigma Chemical, St Louis, MO) was administered i.v. to animals 15 min before the start of the experimental procedure. Fluorescence intensity (excitation wavelength, 420490 nm; emission wavelength, 520 nm) was detected using a silicon-intensified fluorescence camera (model C2400-08: Hamamatsu Photonics, Hamamatsu, Japan). Images were recorded for playback analysis using a videocassette recorder (Figure 1). The fluorescent intensity representing FITCBSA was measured within a defined area (10 x 30 µm) of the venule under study as well as in the adjacent perivascular interstitium. An index of vascular albumin leakage (DPE/DCE) was determined as a ratio using the formula [(interstitial intensity background intensity)/(venular intensity background intensity)] x 100 (maximal value, 100), as previously described.
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Systemic haematocrit measurement
Three small samples of blood obtained from the carotid artery were placed in heparinized capillary tubes (Hemato-Clad Heparinized, Drummond Scientific Company, Broomall, PA), which were spun in a centrifuge (Bilmeter-E, Mochida Phamaceutical, Tokyo, Japan) for 3 min at 7000 g to separate the total blood volume into red blood cell and plasma in order to calculate the haematocrit (Hct).
Statistical analysis
Differences of the data among groups were examined by one-way ANOVA with Fishers multiple comparison test. P-values <0.05 were considered to be significant throughout the current study.
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Results |
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Changes of erythrocyte velocity, vessel diameter and shear rate
Table I shows the shear rate for post-capillary venules in the estrus phase, in the metestrus phase and in the gonadotrophin-treated groups. VR and DV in the microcirculation of the injected groups did not differ significantly. Shear rates in arterioles and venules also did not differ among these groups. The absence of differences among the seven groups in behaviour of erythrocytes permitted consideration of the velocity and density of leukocytes as an index of leukocyte endothelium adhesive interactions.
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Discussion |
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Administration of hMG followed by hCG significantly increased vascular protein leakage within a few hours of the latter injection. After 16 h, rolling velocities of leukocytes in venules were reduced and numbers of leukocytes adherent to endothelium were increased. The results of the present study demonstrate alterations of permeability and interactions between leukocytes and endothelial cells in mesenteric postcapillary venules associated with induction of ovulation. Permeability of mesenteric venules gradually increased after injection of hCG, and leukocyte adhesion rose concurrently.
With regard to increased permeability associated with leukocyte adhesion, most in-vitro and in-vivo studies have demonstrated that leukocyte adhesion, leukocyte emigration and increased protein leakage are closely related. Studies in cat mesentery demonstrated that both platelet-activating factor (PAF) and leukotriene B4 (LTB4) promoted leukocyte adherence in postcapillary venules, but only PAF-induced leukocyte adherence increased vascular protein leakage (Kubes et al., 1991). These investigators proposed that differences in induced oxidant production between these two inflammatory mediators might be related to these distinctive permeability responses. Thus, attachment of leukocytes to the endothelial cells lining the walls of small vessels may not always result in increased microvascular permeability. However, leukocyte adhesion in frog mesentery venules induced by low flow rate was associated with a sustained significant increase in permeability, which differed from the transient increase in permeability that another investigator observed when the venule was exposed to inflammatory mediators (He and Curry, 1994
; He et al., 1996
). A previously reported in-vivo method was used successfully to study leukocyte adhesion and its effect on permeability of the microvasculature. Leukocyte adhesion induced by low flow rate caused a sustained increase in hydraulic conductivity in the venules of frog mesentery. In that system, increasing intracellular cAMP prevented the permeability increase associated with leukocyte adhesion without affecting the adhesion process (He et al., 2000
). These observations indicated that cAMP-dependent mechanisms regulate increases in permeability from inflammatory agents that do not necessarily affect leukocyte adhesion to the venular wall. Thus, interaction between leukocytes and endothelial cells is one of multiple possible causes of increased permeability. In our hMG 54 hCG 16 group, leukocyte rolling velocity and leukocyte adhesion, which are indices of interactions between leukocytes and endothelial cells, were altered significantly. At this time point, microvascular permeability most probably was increased as a result of leukocyte adhesion.
The mechanism underlying the clinical manifestations of OHSS appears to be an increase in capillary permeability of the ovarian vessels and those of other mesothelial surfaces (Mordel et al., 1993; Schenker et al., 1993
; Goldsman et al., 1995
). In this study, we did not examine ovarian albumin leakage, but instead visualized mesenteric postcapillary venules at induction of ovulation. Lowered peripheral vascular resistance, tachycardia and increased cardiac output were observed in 31 consecutive cases of severe OHSS; whether vasodilation preceded or followed any increase in capillary permeability in these patients, or whether the two in fact were related, was unclear (Mathur et al., 1997
). Arteriolar dilation is probably not the sole cause of increased transudation of fluid into the extravascular space at ovulation, since no arteriolar dilation occurred upon induction in our present study.
Soluble forms of the adhesion molecule ICAM-1 assayed in serum have been reported to correlate with biological and clinical features of severe OHSS (Abramov et al., 2001). These endotheliumleukocyte adhesion molecules therefore may be involved in the pathophysiology of the syndrome. Leukocyte-mediated tissue damage is a major component of various human disease processes, including adult respiratory distress syndrome, autoimmune disease, graft rejection and ischaemia reperfusion injury. Endothelial cellleukocyte adhesion molecules have been implicated as mediators in most of these disorders. OHSS may be another entity where adhesion molecules exaggerate leukocyte recruitment and transendothelial leukocyte migration, causing tissue damage and capillary hyperpermeability. ICAM-1 is a member of the immunoglobulin superfamily that is also expressed by vascular endothelial cells. It contains five extracellular immunoglobulin domains that can bind to leukocytes through specific integrins on their surface, mostly
2-integrins. ICAM-1 is expressed in abundance on vascular endothelium after several hours of stimulation by inflammatory cytokines. While endothelial cell surface ICAM-1 appears to contribute to adhesion and transmigration of most leukocyte types through interaction with
2-integrins, neutrophils, monocytes, lymphocytes and natural killer cells all express CD11a/18, which also has been shown to bind to ICAM-1 (Springer et al., 1990
). In the present study, pretreatment with mAb against ICAM-1 significantly inhibited the increase of microvascular permeability and adhesion of leukocytes associated with injection of hMG and hCG. OHSS may be one of several disease entities in which these adhesion molecules cause harmful leukocyte recruitment.
Albumin leakage, but not leukocyte adhesion, was evident at 8 h after injection of hCG, while leukocyte rolling velocity was significantly decreased at 16 h after injection of hCG. Mediators such as histamine, bradykinin and serotonin induce a leukocyte-independent increase in microvascular permeability, while actions of PAF, cytokine-induced neutrophil chemoattractant/growth-related oncogene (CINC/gro), LTB4, formyl-Meth-Leu-Phe (fMLP) as well as more complex models of inflammation such as ischaemiareperfusion injury show a neutrophil-dependent component (Wedmore et al., 1981; Kubes et al., 1996
). Histamine-induced albumin leakage occurs rapidly prior to any changes in leukocyte adhesion. Histamine or other leukocyte-independent mediators may be involved in the early hyperpermeability observed at 8 h after injection of hCG.
Levin et al. (1995) described 22 patients with severe OHSS, five of whom had pleural effusion (23%). Notably, however, capillary accumulation of stimulated leukocytes at physiological shear rates appears to be unique to the pulmonary microcirculation. In other organs such as heart, brain and skeletal muscle, leukocyte entrapment in capillaries occurs only when the microvascular system is exposed to low-shear conditions elicited by haemorrhagic shock or ischaemia. This difference is reflected in observed intravascular events. First, leukocyte rolling, a caterpillar-like movement, is not observed in unstimulated leukocytes within arterioles or venules in the lungs, while the same leukocytes roll and adhere to venules in other organs. This finding indicates that under experimental conditions, transient interactions between leukocytes and endothelium in steady-state lung are quite different from those in other organs. Secondly, blockade of SLeX, a ligand for P-, E- and L-selectins, does not alter the leukocyte behaviour in the lung. Thirdly, when activated, leukocytes were trapped mainly in the pulmonary microvasculature despite the absence of L-selectin (Aoki et al., 1997
). One cause of pleural effusion in OHSS appears to be an interaction between leukocytes and pulmonary vascular endothelium.
In conclusion, our study demonstrated that albumin leakage from mesenteric venules occurs on induction of ovulation by administration of hMG and hCG. Further, albumin leakage is followed by interactions between leukocytes and endothelial cells. Pretreatment with mAb against ICAM-1 significantly inhibited the increase in microvascular permeability and adhesion of leukocytes. ICAM-1 may be important in these leukocyte behaviours and changes in permeability, which are likely to be similar in OHSS.
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Acknowledgement |
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Submitted on December 9, 2002; accepted on February 25, 2003.