Circulating Selectin- and Immunoglobulin-Type Adhesion Molecules in Acute Ischemic Stroke
Background and Purpose Cellular adhesion molecules mediate adhesion between endothelial cells and leukocytes as a precondition for extravasation of leukocytes at sites of tissue injury. The pattern of release of circulating adhesion molecules has been characterized in patients with acute ischemic stroke.
Methods Serum concentrations of soluble selectin-type adhesion molecules (soluble endothelial leukocyte adhesion molecule–1 [sELAM-1], soluble lymph node homing receptor [sL-selectin]) and immunoglobulin-type adhesion molecules (soluble vascular cell adhesion molecule–1 [sVCAM-1], soluble intercellular adhesion molecule–1 [sICAM-1]) were serially determined (at hours 4, 8, and 10 and at days 1, 3, and 5) in 22 patients with acute ischemic stroke. As control subjects, age- and sex-matched individuals with (n=40) and without (n=22) vascular risk factors were studied.
Results We observed increased concentrations of sICAM-1 and decreased levels of sL-selectin in patients with risk factors even in the absence of stroke. Patients with acute stroke had, in addition, an initial transient increase of sELAM-1 and a persistent increase of sVCAM-1.
Conclusions The results suggest a chronic alteration of expression of adhesion molecules sICAM-1 and sL-selectin in subjects with risk factors for atherosclerosis; they also indicate acute changes of levels of sELAM-1 and sVCAM-1 in response to acute ischemic stroke. Determination of soluble adhesion molecules could allow in vivo monitoring of the initial steps of leukocyte-mediated brain damage in acute ischemic stroke.
Recent experimental studies demonstrate massive leukocyte extravasation at sites of cerebral ischemia even within the first hours of disease.1 2 3 4 Leukocytes are now considered to potentiate ischemic neuronal damage by microvasculature obstruction2 5 and generation of neurotoxic substances such as reactive oxygen metabolites, granular enzymes, or toxic cytokines.6 This is corroborated by recent observations that ischemic brain injury is attenuated by rendering the animals leukopenic.3 7 8
Normally, vascular endothelial cells have low adhesiveness for leukocytes. However, when stimulated, for example by cytokines, they express surface adhesion molecules that are responsible for adhesion and activation of leukocytes at sites of tissue irritation and subsequent transendothelial migration of leukocytes into damaged tissue. Selectin-type ELAM-1 and immunoglobulin-type adhesion molecules VCAM-1 and ICAM-1 are located primarily at surfaces of activated endothelial cells and bind leukocytes, whereas L-selectin is constitutively located at the surface of leukocytes and supports their adhesion to activated endothelial cells.9 10 The selectins share a structural domain similar to that described in calcium-dependent vertebrate lectins and they recognize an overlapping set of carbohydrate structures at surfaces of interacting cells. In contrast, the members of the immunoglobulin superfamily, ICAM-1 and VCAM-1, interact with integrins (eg, VLA-4 or LFA-1 and MAC-1) at cellular surfaces.
Soluble isoforms of these adhesion molecules thought to be shed from the surfaces of activated cells can now be quantified in peripheral blood.11 12 13 Increased serum concentrations have been observed in different acute and chronic inflammatory diseases.13 14
The aim of this study was to characterize the pattern of release of these adhesion molecules in patients with acute stroke in comparison to the pattern in age- and sex-matched subjects with and without risk factors for atherosclerosis.
Subjects and Methods
Twenty-two patients admitted within 4 hours after onset of symptoms with acute ischemic stroke were studied (Table 1⇓). Diagnosis was based on history, initial and follow-up neurological examinations, and computed tomography. Ischemia was confined to the carotid arterial territory in 19 patients and to the vertebrobasilar territory in 3 patients. Their demographic characteristics and risk factor profiles are shown in Table 1⇓. One patient died on day 5 and one on day 7.
The diagnostic subgroups were as follows: cardioembolic (n=6), large-vessel occlusive disease (n=6), small-vessel disease (n=3), and unknown or other (n=7). Standard therapy consisted of high-dose (n=14) or low-dose (n=8) heparin. Barthel scores were determined between the second and third week.
As control subjects, 22 healthy people and 40 people with major risk factors for atherosclerosis (smoking, hypertension, diabetes mellitus, or hypercholesterolemia) were studied (Table 1⇑). Age and sex did not significantly differ between the patient and control groups. Exclusion criteria for patients and control subjects were presence of infections, other inflammatory or malignant diseases, conditions associated with tissue injury (eg, stroke, myocardial infarction, or major surgical procedures) within the last year, or immunosuppressive treatment.
Blood Sampling and Quantification of Soluble Adhesion Molecules
Blood was obtained at hours 4, 8, and 10 and at days 1, 3, and 5 after onset of symptoms.
After collection either from peripheral intravenous cannulas or by venipuncture, blood was allowed to clot at room temperature for 1 hour, and after centrifugation the serum was stored at −80°C until it was used. Enzyme immunoassays for sELAM-1 and sL-selectin were purchased from Bender MedSystems. Concentrations of circulating ICAM-1 and VCAM-1 were determined with quantitative enzyme immunoassays (British Biotechnology Products Ltd and T-Cell Diagnostics, Inc, respectively). The intra-assay coefficients of variation for sELAM-1, sL-selectin, sVCAM-1, and sICAM-1 were less than 4.0%, 5.5%, 6.0%, and 3.0%, respectively; the interassay coefficients of variation were 4.5%, 7.6%, 10.2%, and 4.5%, respectively. The lower limits of detection of sELAM-1, sL-selectin, sVCAM-1, and sICAM-1 were 1.6, 0.3, 2.0, and 0.3 ng/mL, respectively.
Results are expressed as mean±SEM. For conservative statistical analysis, the Mann-Whitney U test was used with a Bonferroni correction. Thus, values of P<.05/6 (six being the number of the different study intervals for each patient investigated) were required for differences to be considered significant. For longitudinal analysis, the Friedman two-way ANOVA was used and followed by a Wilcoxon matched-rank test.
Adhesion Molecules in Patients With Vascular Risk Factors
For differentiation between release of adhesion molecules related to acute ischemic stroke and those related to underlying systemic vascular disease, age- and sex-matched subjects with vascular risk factors were included in the study as a control group. Patients with risk factors for atherosclerosis had significantly increased levels of sICAM-1 and significantly decreased levels of sL-selectin, whereas concentrations of sVCAM-1 and sELAM-1 did not significantly differ (Table 2⇓). An increase in the number of risk factors tended to be associated with increased concentrations of sICAM-1 and decreased levels of sL-selectin (Table 2⇓).
Selectin-Type Adhesion Molecules in Acute Stroke
Compared with control subjects with vascular risk factors, patients with acute stroke had significantly increased levels of sELAM-1 at hours 8 and 10 and at day 1, but not later (Fig 1⇓). Also, longitudinal analysis revealed that levels of sELAM-1 at hour 4 were significantly increased compared with those at days 3 (P<.005) and 5 (P<.01).
Levels of sL-selectin in stroke patients did not significantly differ from those in control subjects with risk factors (Fig 1⇑), although a significant decrease at the subacute phase of stroke was observed by longitudinal analysis: concentrations at day 3 were significantly decreased compared with those at hours 4 (P<.005), 8 (P<.01), and 10 (P<.005) and at day 1 (P<.05).
Immunoglobulin-Type Adhesion Molecules in Acute Stroke
Compared with subjects with vascular risk factors, levels of sVCAM-1 were significantly increased in patients with acute stroke (Fig 2⇓). In contrast to the transient increase of sELAM-1, the increase of sVCAM-1 persisted until day 5. Longitudinal analysis showed that initially increased values of sVCAM-1 did not significantly change during the entire study period. Concentrations of sICAM-1, already increased in subjects with vascular risk factors, did not increase further in stroke patients.
Relation of Adhesion Molecules to Age, Sex, Treatment, and Clinical Outcome
Levels of adhesion molecules did not correlate with age or differ with regard to sex in the study groups. Levels in patients with high- or low-dose heparin did not significantly differ. No significant correlation was observed between levels of circulating adhesion molecules and Barthel scores (data not shown).
This study showed that the presence of risk factors for atherosclerosis was already associated with increased serum concentrations of sICAM-1 and decreased levels of sL-selectin, indicating a chronic inflammatory endothelial activation. In addition, patients with acute ischemic stroke exhibited a transient increase of concentrations of sELAM-1 until day 1 and a persisting increase in levels of sVCAM-1 for at least 5 days.
The increased levels of sICAM-1 in subjects with risk factors and stroke are consistent with a previous demonstration of an expression of ICAM-1 mRNA in brain microvascular endothelial cells during hypoxia/reoxygenation20 as well as with the observation of upregulation of ICAM-1 in microvessels in the ischemic zone of focal brain ischemia in experimental21 or postmortem22 studies. However, it must be noted that in our study sICAM-1, in contrast to sELAM-1 or sVCAM-1, was already increased in subjects with vascular risk factors in the absence of acute stroke. This is consistent with earlier reports of elevated levels of circulating adhesion molecules in diabetic patients12 16 and with observations of a massive upregulation of ICAM-1 in contrast to an only modest expression of VCAM-1 and ELAM-1 in atherosclerotic vessels.17
Because levels of circulating adhesion molecules may represent the net sum of their shedding and their clearance by binding to counterreceptors, what is measured in the circulation may not include the portion that is bound to counterreceptors. Therefore, the decrease of the leukocyte-derived sL-selectin in subjects with vascular risk factors may be explained by its binding to upregulated endothelial counterreceptors (eg, oligosaccharides) in chronic and acute endothelial inflammation.
Patients with acute stroke showed changes in concentration of sICAM-1 and sL-selectin similar to those observed in subjects with vascular risk factors. However, sELAM-1 and sVCAM-1 were, in addition, significantly increased, which may reflect their acute upregulation and shedding at sites of ischemic cerebral tissue lesion. These adhesion molecules mediate adhesion, activation, and subsequent passage of leukocytes into injured tissue.9 13 The early increase of the endothelial-specific adhesion molecules sELAM-1 and sVCAM-1 is in accordance with recent observations of the leukocyte extravasation as early as 30 to 60 minutes in microvessels after experimental middle cerebral artery occlusion.1 2 3 4 17 These cells play a key role in prolonged ischemic brain damage by microvasculature obstruction2 5 and release of neurotoxic substances such as reactive oxygen metabolites, toxic enzymes, or cytokines.6
A characteristic temporal profile of release of adhesion molecules evolved: initially, sELAM-1 was increased concomitantly with sVCAM-1, whereas in a later phase levels of sELAM-1 returned to normal values despite persistence of increased levels of sVCAM-1. Interestingly, results of recent in vitro studies showed that ELAM-1 is similarly upregulated only transiently during the first hours, whereas immunoglobulin-type adhesion molecules are persistently expressed at cellular surfaces for at least 72 hours after cytokine or thrombin stimulation.18 19 This corresponds with current concepts of the initial steps of inflammation; therefore, selectins are considered to sustain the phenomenon of margination and the initial light attachment of circulating leukocytes to activated microvascular endothelium. The immunoglobulin-type adhesion molecules are particularly important for the subsequent firm attachment and transendothelial migration into the surrounding tissue.9 13
Until now, little has been known about the mechanisms of shedding or the metabolic fate of these molecules. However, in vitro studies show that selectin- and immunoglobulin-type adhesion molecules are rapidly lost from the surface of endothelial cells or leukocytes after experimental stimulation.14 A possible link between ischemic brain injury and the expression of these substances may represent the release of proinflammatory cytokines such as tumor necrosis factor–α or interleukin-1ß, which have been detected in different types of tissue damage,23 including focal cerebral ischemia,24 and have been shown to induce upregulation and shedding of adhesion molecules.18
Although levels of adhesion molecules did not significantly differ between patients with high- or low-dose heparin treatment, and patients did not have severe hepatic or renal disorders, further studies should clarify the effect of heparin treatment and metabolism on concentrations of these substances.
In conclusion, the results suggest a chronic endothelial inflammation already in subjects with risk factors for atherosclerosis, as well as acute changes of expression of adhesion molecules in acute stroke consistent with current concepts of leukocyte adhesion at hypoxically injured cerebral tissues. Determination of these molecules could allow in vivo monitoring of key events of leukocyte-mediated damage in ischemia and reperfusion.
Selected Abbreviations and Acronyms
|sELAM-1||=||soluble endothelial leukocyte adhesion molecule–1|
|sICAM-1||=||soluble intercellular adhesion molecule–1|
|sL-selectin||=||soluble lymph node homing receptor|
|sVCAM-1||=||soluble vascular cell adhesion molecule–1|
The authors wish to thank I. Poltersdorf for excellent technical assistance.
- Received December 8, 1994.
- Revision received April 19, 1995.
- Accepted April 28, 1995.
- Copyright © 1995 by American Heart Association
Hallenbeck JM, Dutka AJ, Tanishima T, Kochanek PM, Kumaroo KK, Thompson CB, Obrenovitch TP, Contreras TJ. Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke. 1986;17:246-253.
del Zoppo GJ, Schmid-Schönbein GW, Mori E, Copeland BR, Chang C-M. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke. 1991;22:1276-1283.
Matsuo Y, Onodera H, Shiga Y, Nakamura M, Ninomiya M, Kihara T, Kogure K. Correlation between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in the rat: effects of neutrophil depletion. Stroke. 1994;25:1469-1475.
Bednar MM, Raymond S, McAuliffe T, Lodge PA, Gross CE. The role of neutrophils and platelets in a rabbit model of thromboembolic stroke. Stroke. 1991;22:44-50.
Chen H, Chopp M, Bodzin G. Neutropenia reduces the volume of cerebral infarct after transient middle cerebral artery occlusion in the rat. Neurosci Res Commun. 1992;11:93-99.
Williams TJ, Hellewell PG. Endothelial cell biology: adhesion molecules involved in the microvascular inflammatory response. Am Rev Respir Dis. 1992;146:45-50.
Springer TA. Adhesion receptors of the immune system. Nature. 1990;34:425-434.
Rothlein R, Mainolfi EA, Czajkowski M, Marlin SD. A form of circulating ICAM-1 in human serum. J Immunol. 1991;147:3788-3793.
Newman W, Beall LD, Carson CW, Hunder GG, Graben N, Randhawa ZI, Gopal TV, Wiener-Kronisch J, Matthay MA. Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in serum of patients with septic shock. J Immunol. 1993;150:644-654.
Davies MJ, Gordon JL, Hearing AJ, Rigott R, Woolf N, Katz D, Kyriakopoulos A. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol. 1993;17:223-229.
Sugama Y, Tiruppathi C, Janakidevi K, Andersen TT, Fenton JW II, Malik AB. Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion. J Cell Biol. 1992;11:935-944.
Hess DC, Zhao W, Carroll J, McEachin M, Buchanan K. Increased expression of ICAM-1 during reoxygenation in brain endothelial cells. Stroke. 1994;25:1463-1468.
Okada Y, Copeland BR, Mori E, Tung MM, Thomas WS, del Zoppo G. P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion. Stroke. 1994;25:202-211.
Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ. Tumor necrosis factor-α expression in ischemic neurons. Stroke. 1994;25:1481-1488.