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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:258-268.)
© 1995 American Heart Association, Inc.

Articles

Venous Thrombosis–Associated Inflammation and Attenuation With Neutralizing Antibodies to Cytokines and Adhesion Molecules

Thomas W. Wakefield; Robert M. Strieter; Carol A. Wilke; Amy M. Kadell; Shirley K. Wrobleski; Marie D. Burdick; Regina Schmidt; Steven L. Kunkel; Lazar J. Greenfield

From the Section of Vascular Surgery and Jobst Vascular Research Laboratory, Department of Surgery (T.W.W., A.M.K., S.K.W., R.S., L.J.G.); the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine (R.M.S., C.A.W., M.D.B); and the Department of Pathology (S.L.K.), University of Michigan Medical Center, Ann Arbor.

Correspondence to Thomas W. Wakefield, MD, 2210D THCC, University of Michigan Medical Center, 1500 E Medical Center Dr, Ann Arbor, MI 48109-0329.

	Abstract

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Abstract Thrombosis and inflammation are closely related. However, the response of the vein wall to venous thrombosis has been poorly documented. This study examines the hypothesis that venous thrombosis is associated with an inflammatory response in the vein wall. In a rat model of inferior vena caval thrombosis, vein wall was temporally examined for inflammation by assessment of histopathology, leukocyte morphometrics, and cytokine levels. Animals were killed 1 hour and 1, 3, and 6 days after thrombus induction. Our findings demonstrated an early (day 1) neutrophil infiltration into the vein wall followed by a later (days 3 and 6) monocyte/macrophage and lymphocyte response. Cytokines were elevated only under conditions of venous thrombosis. Levels of epithelial neutrophil activating protein–78 (ENA-78), tumor necrosis factor– (TNF), interleukin-6, and JE/monocyte chemoattractant protein–1 (JE/MCP-1) increased over the 6-day period, while macrophage inflammatory protein-1 (MIP-1) peaked at day 3 after thrombus induction. Additionally, rats were passively immunized with neutralizing antibodies to TNF, ENA-78, MIP-1, JE/MCP-1, intercellular adhesion molecule–1 (ICAM-1), and CD18 compared with control antibodies. The most effective antibody early after thrombus induction for attenuating vein wall neutrophil extravasation was anti-TNF (P<.01). The monocyte/macrophage extravasation was inhibited most by anti–ICAM-1 followed by anti-TNF (P<.01). These findings demonstrate that venous thrombosis is associated with significant vein wall inflammation that is partially inhibited by neutralizing antibodies to cytokines and adhesion molecules.

Key Words: venous thrombosis • inflammation • antibodies • neutrophils • monocytes/macrophages

	Introduction

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Venous thromboembolism is a national health problem, occurring with an annual incidence of 250 000 cases.¹ It has been estimated that deep venous thrombosis and pulmonary embolism are associated with 300 000 to 600 000 hospitalizations and as many as 50 000 deaths per year.¹ The sequela of venous thrombosis, chronic venous insufficiency, affects up to 400 000 to 500 000 patients with skin ulceration and 6 to 7 million patients with skin stasis changes.¹ The prevalence of leg ulceration associated with chronic venous insufficiency is 5 of 1000 individuals in the United States older than 20 years of age.¹ Approximately 1% of adults (3% to 4% of those over 65 years of age) once had or now have an ulcer of the leg from a chronic venous cause.¹ Significant morbidity is associated with venous thrombosis in as many as 90% of patients suffering from significant disability at 2- to 5-year follow-up intervals.² Venous reflux develops progressively over time after thrombosis, with more than 67% of involved limbs developing venous reflux after 1 year.³ However, factors other than incomplete lysis of thrombus are involved in this pathogenesis.⁴ We hypothesize that chronic venous insufficiency is a result of the interrelation between thrombosis and inflammation (phlebitis) that results in destruction of venous valves and chronic vein wall changes that lead to venous reflux and the syndrome of chronic venous insufficiency. For example, tumor necrosis factor– (TNF), a polypeptide inflammatory cytokine generated during sepsis, downregulates natural anticoagulant mechanisms such as proteins C and S and the fibrinolytic system while inducing expression of procoagulant tissue factor on the surface of vascular endothelium supporting thrombosis.^{5 6 7 8 9 10 11} We used a reproducible model of venous thrombosis in the rat^{12 13 14} involving venous stasis to further characterize the relation between thrombosis and the inflammatory response leading to phlebitis. In this model, we investigated temporal leukocyte extravasation in response to venous thrombosis by morphometric analysis. These events were correlated with the expression of proinflammatory cytokines. Furthermore, we assessed these events under conditions in which the animals received passive immunization with neutralizing antibodies to TNF, epithelial neutrophil activating protein–78 (ENA-78), human macrophage inflammatory protein-1 (MIP-1), JE/monocyte chemoattractant protein–1 (JE/MCP-1), intercellular adhesion molecule–1 (ICAM-1), and CD18 compared with control antibodies. We found that anti-TNF and anti–ICAM-1 were the most effective antibodies for attenuating the inflammatory events within the vein wall.

	Methods

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Animal Model and Protocol
We investigated 90 Sprague-Dawley rats in this study, 48 rats in group 1 and 42 rats in group 2. We anesthetized 20 rats in group 1 with isoflurane and induced inferior vena caval (IVC) thrombosis by IVC ligation just below the left renal vein.^{12 13 14} At 1 hour and then at days 1, 3, and 6 after thrombus initiation (n=5 at each time point), rats were killed, and portions of the IVC were analyzed histologically and morphometrically for leukocyte trafficking and immunohistochemical staining for the following cytokines: TNF, interleukin-6 (IL-6), the C-X-C chemokine (ENA-78), and the C-C chemokines (JE/MCP-1 and MIP-1). Additionally, 4 animals each (12 total in group 1) were killed at days 1, 3, and 6 after thrombus induction for tissue extraction and enzyme-linked immunosorbent assay (ELISA) determination of the IVC wall for the above cytokines. In group 1, 12 rats underwent sham IVC dissection without ligation; they were killed at days 1, 3, and 6 (n=4 each) for histopathology, leukocyte morphometrics, immunohistochemical staining, and cytokine measurement by specific ELISA. Additionally, 4 animals in group 1 underwent abdominal exploration for IVC harvest without IVC thrombosis for baseline analysis of histopathology, leukocyte morphometrics, immunohistochemical cytokine localization, and cytokine measurement by specific ELISA. At the time of death, 28 of 32 IVCs were found thrombosed, including all 12 IVCs removed for ELISA tissue analysis and 16 of 20 IVCs removed for histopathology, morphometrics, and immunohistochemical staining. All 12 sham and 4 baseline control IVCs were patent.

Forty-two rats (group 2) underwent treatment with various polyclonal rabbit anti-murine antibodies or monoclonal murine anti-rat antibodies in cohort groups of 3 animals each with death either 1 or 6 days after thrombus induction. Polyclonal rabbit anti-murine cytokine (TNF, ENA-78, MIP-1, and JE/MCP-1) serum was administered daily (1 mL/d) into the tail vein. These antibodies are specific and cross-react only with the specific cytokine in question.^{15 16 17 18} These antibodies were produced by first administering TNF, ENA-78, MIP-1, and JE/MCP-1 in multiple intradermal injections of 20 µg (total) emulsified with Freund's complete adjuvant to rabbits. This procedure was repeated in 10 days with 20 µg of TNF, ENA-78, MIP-1, or JE/MCP-1 emulsified in Freund's incomplete adjuvant. Rabbits were bled 10 days later with the antiserum, then isolated, and heat inactivated. A direct ELISA was then performed to establish the efficacy of the rabbit antiserum to detect the cytokines, and no cross-reactivity to other cytokines was discovered. These antibodies were endotoxin-free as determined with a standard Limulus assay.¹⁹ Preimmune rabbit serum was used as control. Monoclonal murine anti-rat antibodies included antibodies to ICAM-1 (1A29, 2 mg/kg given daily into the tail vein) and CD18 (CL-26, 1 mg/kg given daily into the tail vein). The 1A29 is an anti–ICAM-1 IgG1 antibody that reacts with an 85- to 89-kD epitope present on cytokine-activated rat endothelial cells,^{20 21 22} while CL-26 is an IgG1 antibody that reacts with a 95-kD protein band on rat neutrophils and spleen cells consistent with CD18.^{23 24} These doses have been found to attenuate ICAM-1 and CD18 responses in the rat.²⁵ These monoclonal antibodies were also endotoxin-free by Limulus assay. At the time of death, the animal's IVC was harvested for histopathology, morphometric analysis, and tissue extraction for cytokine levels by specific ELISA. Immunohistochemistry was possible only for those animals given monoclonal murine anti-rat antibodies directed against ICAM-1 and CD18 because of interference between the rabbit anti-cytokine administered serum and the staining procedure.

Histopathology and Morphometrics
Leukocyte extravasation and trafficking were assessed with histological evaluation (by a pathologist) and morphometric analysis. Morphometrics was performed by counting three levels of tissue, five sections per level (high-power field [HPF], x1000) after the slides were stained with hematoxylin and eosin in standard fashion. Each vein wall was evaluated and noted for the number of cells in the five HPFs. For morphometry, the vein wall studied included the intima, media, and adventitia. Analysis began at the thrombus-vein wall interface and extended the width of a high-power field. Cells were identified as neutrophils, monocytes/macrophages, or lymphocytes on the basis of standard morphological criteria, including nuclear size, cytoplasmic content, and cell size.

Immunohistochemical Analysis
Immunohistochemistry was used to determine cellular localization of cytokine antigen in the vein wall. Paraffin-embedded tissues were sectioned, deparaffinized with xylene, and rehydrated with serial dilutions of ethanol. On each slide, a tissue section exposed to antibody (experimental) and a section exposed only to normal rabbit serum (control) were analyzed. Immunohistochemical staining was performed with a biotin streptavidin–amplified detection system with streptavidin-conjugated alkaline phosphatase (Biogenex Laboratories).²⁶ Tissue was blocked for nonspecific binding sites with normal goat serum. Preimmune rabbit serum or rabbit polyclonal anti–murine cytokine antibodies at concentrations between 1:250 and 1:500 were then applied to the tissue sections and allowed to incubate for 90 minutes. Slides were then washed, followed by the application of a goat anti-rabbit IgG biotinylated antibody at a concentration of 1:35, followed by incubation for 60 minutes at 37°C. The tissue was then washed and incubated with streptavidin-conjugated alkaline phosphatase at a 1:35 concentration for 40 minutes at 37°C. After the tissue was washed again, the substrate-buffered Naphthol solution was applied, and the reaction was extinguished when color began to develop on the control section. The tissue was then counterstained with 0.1% Mayer's hematoxylin. Light microscopy assessed specific cell-associated staining in the vein wall, comparing the experimental and control sections.

Tissue Preparation and Cytokine ELISAs
This technique allowed quantification of cytokines in tissue as previously described.²⁷ Briefly, tissue homogenization of vein wall segments was performed in lysis buffer (1x phosphate-buffered saline [PBS] with 2 mmol/L phenylmethylsulfonyl fluoride and 1 µg/mL of each of the following: antipan, aprotinin, leupeptin, pepstatin A [Sigma Chemical Co]) with a hand-held homogenizer followed by sonication on ice for 30 seconds. The sonicated tissue was then centrifuged at 1500g for 10 minutes, followed by filtration through a 1.2-µm syringe filter. Immunoreactive cytokine levels were quantified with a double-ligand method as previously documented.²⁷ Flat-bottomed 96-well microtiter plates were coated with 50 µL per well specific rabbit anti-cytokine antibody (1 ng/µL in 0.6 mol/L NaCl, 0.26 mol/L H₃PO₄, and 0.08N NaOH, pH 9.6) for 16 hours at 4°C and then washed. Microtiter plate nonspecific binding sites were blocked with 2% bovine serum albumin in PBS and incubated for 60 minutes at 37°C. Plates were then rinsed three times, and diluted (1:5 and 1:10) specimen (50 µL) in duplicate was added, followed by incubation for 60 minutes at 37°C. Plates were then washed three times, followed by the addition of 50 µg per well biotinylated rabbit anti-cytokine antibody (3.5 µg/mL in PBS, pH 7.5, 0.05% Tween-20, and 2% fetal calf serum), and were incubated for 45 minutes at 37°C. These secondary antibodies were specific for the cytokine being tested with no cross-reactivity to other cytokines found. Plates were again washed three times, streptavidin-peroxidase conjugate (1:5000) was added (100 µL per well, Bio-Rad Laboratories), and the plates were incubated for 30 minutes at 37°C. Plates were again washed, and the chromogen substrate o-phenylenediamine dihydrochloride (100 µL per well) was added. Plates were then incubated at room temperature to the desired extinction, and the reaction was terminated with 50 µL per well of 3 mol/L H₂SO₄ solution. Plates were read at 490 nm in an ELISA plate reader. Standards were 1/2 log dilutions of the cytokines from 1 pg/mL to 100 ng/mL. The sensitivity of the ELISAs was 50 pg/mL. Administration of rabbit anti-cytokine antibody to the rats has been found not to interfere with the performance of these ELISA measurements. This was confirmed previously with a bioassay for TNF using WEHI 164 cells, with declines in functional TNF by as much as 98% when rat lung homogenates were preincubated with neutralizing rabbit anti-murine TNF- serum.²⁸

Statistical Evaluation and Animal Use
Statistical evaluation included mean±SEM and unpaired Student's t tests where appropriate. All animals used in this study were housed and cared for in the University of Michigan Unit for Laboratory Animal Medicine under the direction of a veterinarian according to the Principles of Laboratory Animal Care (National Society for Medical Research) and Guide for the Care and Use of Laboratory Animals (National Institutes of Health [NIH] Publication No. 86-23, revised 1985).

	Results

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Vein Wall Studies (Group 1)
Histological Evaluation
At baseline, few inflammatory cells were noted in the vein wall (Fig 1A). At 1 hour after thrombus induction, neutrophils began to line up at the thrombus–vein wall interface, but the wall remained relatively devoid of an inflammatory cell influx (Fig 1B). At day 1, neutrophil presence in the vein wall was a significant finding, with these cells entering the wall from both the luminal and adventitial sides of the wall; neutrophils appeared to predominate on the adventitial side of the vein wall (Fig 1C). At day 3, neutrophils, monocytes/macrophages, and lymphocytes were seen, along with prominent fibroblast-type cells attempting to "organize" thrombus and vein wall (Fig 1D). At day 6, the thrombus was clearly beginning to organize from the thrombus–vein wall interface, with the definition of this interface becoming indistinct (Fig 1E). The fibroblast-type cells were becoming less prominent at this later time point. In the sham- dissected veins, no similar luminal- or adventitial-oriented inflammatory response was noted.

View larger version (2K): [in this window] [in a new window]   Figure 1. Photomicrographs showing (A) few inflammatory cells noted in the vein wall at baseline; (B) 1 hour after thrombus induction, neutrophils line up along the thrombus–vein wall interface (open arrows); (C) day 1 after thrombus induction, neutrophils dominate the morphological picture, especially in the adventitial portion of the vein wall; (D) day 3 after thrombus induction, a mixture of neutrophils, monocytes/macrophages, and lymphocytes is noted with the development of larger, darkly staining fibroblast-type cells in the vein wall; and (E) day 6 after thrombus induction, the thrombus is showing signs of organization from the thrombus–vein wall interface (arrows) inward, with this interface becoming indistinct, the fibroblast-type cells becoming less prominent, and the cellular infiltrate composed of nonneutrophilic cells. F, Graph shows change in inflammatory cell count over time. T indicates thrombus; w, vein wall; and a, adventitia. (Hematoxylin and eosin, original magnification x400.)

Vein Wall Leukocyte Morphometric Analysis During Venous Thrombosis
At baseline, only 25±5 leukocytes per vein wall (5 HPFs) were noted (Table 1). However, 1 hour after thrombus induction, 34±5 leukocytes per 5 HPFs were noted, with an absolute leukocyte differential of 5±1 neutrophils, 10±2 monocytes, and 19±3 lymphocytes. By day 1, the cell count increased to 90±7 per 5 HPFs, with 58±7 neutrophils, 19±1 monocytes/macrophages, and 13±1 lymphocytes (Fig 1F). At day 1, in the sham dissected samples, the cell count was 34±2 per 5 HPFs, with 8±2 neutrophils, 23±0 monocytes, and 3±0 lymphocytes (Table 1). At day 3, the cell count in the thrombosed vein wall was 100±4 leukocytes, with a significant increase in monocytes/macrophages to 40±3, while neutrophils decreased to 40±5 and lymphocytes increased to 20±5 (Fig 1F). In the sham animals at day 3, the leukocyte count was 33±2 per 5 HPFs, represented by 21±1 monocytes, 6±1 neutrophils, and 6±0 lymphocytes (Table 1). By day 6, leukocyte infiltration had decreased to 83±2 cells per 5 HPFs, and neutrophils and mononuclear phagocytes declined to 15±4 and 30±1 cells, respectively. However, lymphocytes increased to 38±3 cells per 5 HPFs (Fig 1F). At day 6, in the sham animals, the total leukocyte count was only 37±2 cells per 5 HPFs, reflecting 9±1 monocytes, 6±1 neutrophils, and 22±2 lymphocytes (Table 1). Significant differences in neutrophil and monocyte elevations compared with baseline were noted beginning 1 day after thrombus induction. In a similar fashion, significant differences in total leukocyte count and differential cell counts existed between sham animal dissected vein walls and thrombosed vein walls (Table 1).

View this table: [in this window] [in a new window]   Table 1. Morphometric Analysis (Cells per 5 High-Power Fields) of Inflammatory Cells in the Thrombosed Vein Walls Indexed to the Time From Clot Induction

Cytokine Expression Within the IVC During Thrombosis
To quantify the temporal expression of cytokines in the vein wall during thrombosis, we measured vein wall cytokine content on days 1, 3, and 6 after thrombus formation. We found that TNF significantly increased from baseline (<50 pg/mL) to 127±46 pg/mL (P<.05) at day 1, rising to 341±55 pg/mL (P<.01) at day 3 and peaking at day 6 (610±212 pg/mL, P<.05, Table 2). In those animals in which the IVC was ligated but had no thrombus formation, TNF levels were <50, <50, <50, and 170 pg/mL at baseline and days 1, 3, and 6, respectively, after laparotomy. In the sham animal IVCs, no TNF was detected at days 1, 3, and 6 after sham dissection. ENA-78 increased from baseline at day 3 (175±81 pg/mL) and became significantly elevated at day 6 (370±150 pg/mL, P<.05). MIP-1 was elevated at day 3 (105±45 pg/mL), declining at day 6 to <50 pg/mL. IL-6 levels became elevated from baseline on days 3 (328±187 pg/mL) and 6 (445±243 pg/mL), while JE/MCP-1 was significantly elevated from baseline at days 1 (2372±650 pg/mL, P<.05) and 3 (3198±704 pg/mL, P<.01) and peaked at day 6 (12 914±7073 pg/mL). The corresponding values for the sham-operated animals were consistently less than those for animals with IVC thrombosis. No detectable cytokine, other than a minimal elevation in ENA-78 at day 6 (70±34 pg/mL), was seen. Statistically significant differences between the sham-dissected vein segments and the thrombosed vein segments were noted for TNF (days 1, 3, and 6) and JE/MCP-1 (days 1 and 3).

View this table: [in this window] [in a new window]   Table 2. ELISA Measurements for Cytokines and Chemokines in the Thrombosed Vein Walls Indexed to the Time From Clot Induction for All Time Points Including Baseline

Immunolocalization of Cytokines During IVC Thrombosis
Significant cytokine immunolocalization was present only in the context of venous thrombosis (Fig 2A through 2F). In the first hour after ligation, neutrophils appeared to be the predominant cellular source of TNF within the vein wall and thrombus. By day 1 after ligation, ENA-78 was predominantly associated with neutrophils within the thrombus itself and those that had extravasated into the vein wall. By day 3, MIP-1 was markedly present in both neutrophils, monocytes/macrophages, and smooth muscle cells of the vein wall. By day 6, JE/MCP-1 and IL-6 were the predominantly expressed cytokines within the vein wall. IL-6 was derived from neutrophils, monocytes/macrophages, and vein wall smooth muscle cells.

View larger version (2K): [in this window] [in a new window]   Figure 2. Photomicrographs showing (A) tumor necrosis factor– production from neutrophils in the thrombus and vein wall at day 1 after thrombus induction; (B) epithelial neutrophil activating protein–1 production from primarily neutrophils in the thrombus at day 1 after thrombus induction; (C) macrophage inflammatory protein-1 (MIP-1) production from neutrophils and monocytes in the thrombus and vein wall cells at day 3 after thrombus induction (note the granular staining indicative of MIP-1 protein production); (D) extensive JE/monocyte chemoattractant protein–1 (JE/MCP-1) staining in the vein wall at day 6 after thrombus induction (JE/MCP-1 is known to be taken up by red blood cells); (E) interleukin-6 production at day 6 from inflammatory cells and modified vein wall cells; and (F) no cytokines present in this MIP-1 stain at day 3 after thrombus induction in an animal without clot formation despite ligature placement. T indicates thrombus; w, vein wall; and arrows, thrombus–vein wall interface. (Hematoxylin and eosin, original magnification x1000.)

Passive Immunization Studies (Group 2)
Leukocyte Extravasation in Relation to Thrombosis
To establish whether the expression of the above cytokines was associated with vein wall inflammation, passive immunization was performed with various neutralizing antibodies. At day 1 after thrombus induction, the most effective antibody was anti-TNF, decreasing the total inflammatory cell count from 98±5 (control serum) to 80±8 per 5 HPFs, while the other antibodies were less effective at this time point (Table 3). Importantly, neutrophils in the vein wall were significantly decreased from 63±0 with control serum to 36±1 with anti-TNF serum (P<.01, Fig 3). The percentage of neutrophils was decreased from 64% with control serum to 45% with anti-TNF. The order of effectiveness in limiting neutrophil migration was anti-TNF>>anti–ENA-78 compared with control serum. Antibodies to MIP-1, CD18, JE/MCP-1, and ICAM-1 were not effective in preventing neutrophil extravasation. Neutralizing antibody to ICAM-1 attenuated neutrophil extravasation at the thrombus–vein wall interface compared with neutralizing antibody to CD18, although the neutrophils tended to localize in the adventitial portion of the vein wall (Fig 4). At day 6 after thrombus formation, the most effective neutralizing antibody for attenuating total leukocyte extravasation was the antibody to ICAM-1, decreasing the leukocyte count from 55±1 cells (control serum) to 41±2 cells per 5 HPFs (anti–ICAM-1, P<.01, Fig 3). Monocytes were also markedly inhibited by neutralizing antibody to ICAM-1, from 34±1 cells (control serum) to 8±0 cells per 5 HPFs (anti–ICAM-1, P<.01), followed by neutralizing antibody to TNF, which decreased monocyte extravasation to 19±1 cells (P<.01, Table 3). Monocyte percentage decrease was from 62% for control serum to 20% for anti–ICAM-1 and 34% for anti-TNF. The order of effectiveness in limiting monocytes/macrophage extravasation was anti–ICAM-1>>anti-TNF>anti–JE/MCP-1>anti-CD18>anti–ENA-78 and anti–MIP-1 compared with control serum.

View this table: [in this window] [in a new window]   Table 3. Morphometric Analysis (Cells per 5 High-Power Fields) of Inflammatory Cells in Vein Walls Indexed to the Time From Clot Induction (1 or 6 Days) in Animals Treated With Various Monoclonal and Polyclonal Antibodies

View larger version (2K): [in this window] [in a new window]   Figure 3. Photomicrographs showing neutrophil extravasation into the vein wall in an animal treated with control serum (A) and antibody to tissue necrosis factor– (B) at day 1 after thrombus induction. Neutrophils are extravasating into the vein wall from both the luminal and adventitial portions of the vein wall in the control serum–treated animal. The monocyte/macrophage extravasates into the vein wall in an animal treated with control serum (C) and antibody to intercellular adhesion molecule–1 (D) at day 6 after thrombus induction. In the control animal, the thrombus–vein wall interface can no longer be identified. T indicates thrombus; w, vein wall; and arrows, thrombus–vein wall interface. (Hematoxylin and eosin, original magnification x400.)

View larger version (2K): [in this window] [in a new window]   Figure 4. Photomicrographs showing macrophage inflammatory protein–1 stain (day 1 after thrombus induction) in an animal treated with antibody to intercellular adhesion molecule–1 (ICAM-1) (A) and CD18 (B). Note the attenuation of activated neutrophils (open arrows) at the thrombus–vein wall interface (arrow) in the animal treated with antibody to ICAM-1 vs antibody to CD18 in these two examples. T indicates thrombus; w, vein wall. (Original magnification x1000.)

Cytokine Expression Within the Vein Wall in Relation to Thrombus
At day 1 after thrombus induction, TNF levels were inhibited by antibody to TNF (127±46 decreased to <50 pg/mL). At day 6 after thrombus initiation, antibody to TNF remained moderately effective at decreasing the amount of TNF released (610±212 to 297±91 pg/mL), while antibody to JE/MCP-1 was very effective in reducing JE/MCP-1 (12 914±7073 to 3392±1281 pg/mL). Antibody to ENA-78 also was effective in reducing the amount of ENA-78 released (370±150 to <50 pg/mL). Antibody to ICAM-1 limited TNF and JE/MCP-1 production at day 1 (TNF, 127±46 to <50 pg/mL; JE/MCP-1, 2372±650 to 88±73 pg/mL, P<.05) and day 6 (TNF, 610±212 to 144±68 pg/mL; JE/MCP-1, 12 914±7073 to 165±119 pg/mL) after thrombus induction, suggesting a leukocyte cellular source for these cytokines. A similar reduction was noted for antibody to CD18 for TNF and JE/MCP-1 (day 1: TNF, 127±46 to <50 pg/mL; JE/MCP-1, 2372±650 to <50 pg/mL, P<.05; day 6: TNF, 610±212 to 309±76 pg/mL; JE/MCP-1, 12 914±7073 to 2284±2053 pg/mL, respectively). Statistically significant differences were not found for all these reductions because of the variability in the measurements.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our findings demonstrate an important temporal relation between venous thrombosis and inflammation of the vein wall. Our data suggest that early after IVC thrombosis (day 1), a large influx of neutrophils occurs into the vein wall, and as the thrombus matures, neutrophils are joined by monocytes/macrophages as the predominant leukocytes. At later times (day 6), monocytes/macrophages and lymphocytes are the predominant leukocyte subpopulation. The sham-operated animals and the four animals undergoing ligation without thrombus formation clearly demonstrated the important relation between thrombosis and the inflammatory response. Without thrombus, the inflammatory response does not occur. TNF, the cytokine expressed earliest in the vein wall, appeared to play a pivotal role in mediating the early inflammatory response and extravasation of neutrophils into the vein wall. This was confirmed by the results of passive immunization with antibodies to TNF. At later times, cytokines associated with both neutrophils and monocytes/macrophages, such as MIP-1 and JE/MCP-1, contributed to the monocyte/macrophage infiltration. However, our neutralizing antibody studies also suggest that TNF is an important mediator of the later inflammatory response (day 6). ELISA measurements in the vein walls for those animals treated with appropriate anti-cytokine and anti-adhesion molecules confirm the ability of these antibodies to target and inhibit their specific receptors. Furthermore, the importance of leukocyte adhesion events at the later time points of inflammation was demonstrated with passive immunization with neutralizing antibodies to ICAM-1, which resulted in attenuation of monocyte/macrophage extravasation.

Leukocytes appeared to initially align themselves at the thrombus–vein wall interface (Fig 1B). Extravasation into the vein wall could then have occurred as a result of the generation of a chemokine gradient established in the vein wall,²⁹ from both the luminal and adventitial sides through the vasa vasorum. In fact, extravasation from the adventitial side (Fig 3A) is probably more important in our model in which thrombus forms rapidly and no blood flows past the area of thrombosis. In a primate model of pure stasis-induced vena caval thrombosis, a similar inflammatory response was demonstrated, including the presence of peri-IVC inflammatory enhancement on magnetic resonance venography over the same time course as the present study, suggesting the importance of the thrombus to the generation of inflammation (T.W.W. et al, unpublished data, 1994). Such an inflammatory response may play a major role in the detrimental changes documented in the vein wall and its associated valves after venous thrombosis. Control of inflammation may limit the damage to the vein wall from the inflammatory response induced by the thrombotic stimulus, resulting in a reduction of further thrombosis. In fact, neutralizing antibodies to cytokines and adhesion molecules were effective in the present study in limiting both the early neutrophil and later monocyte/macrophage response.

A model for the interaction between thrombosis and inflammation was proposed previously.³⁰ Concomitant with thrombosis, leukocytes migrate through the thrombus–vein wall interface or through the vasa vasorum and enter the vein wall in significant numbers. Neutrophils have been found to be the first leukocyte to adhere to the endothelium in a model of stasis-induced venous thrombosis in the cat, and the presence of these neutrophils was associated with clot formation.³¹ Neutrophils were found not only to adhere to the endothelium but also to undergo transendothelial migration, leading to endothelial cell sloughing and exposure of the basement membrane. This provided a thrombogenic surface for further thrombosis. The extravasation of neutrophils and later monocytes/macrophages and lymphocytes into the vein wall depends on the establishment of a chemotactic gradient. Our results support this notion and the concept that the early inflammatory response is localized to the adventitia in addition to the region of the thrombus–vein wall interface. Thus, in the context of thrombosis, leukocytes infiltrate the vein wall from both the "outside in" (vasa vasorum) and "inside out" (vascular lumen).

Two families of chemotactic cytokines have been identified that appear to have proinflammatory and reparative activities.^{32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61} In both families, these cytokines, in their monomeric forms, are all less than 10 kD and are characteristically basic heparin-binding proteins. They display four highly conserved cysteine amino acid residues. In one family, the first two cysteine amino acid residues are separated by one nonconserved amino acid. In general, these cytokines appear to have specific chemotactic activity for neutrophils. Because of their chemotactic properties and the presence of the C-X-C cysteine motif, these cytokines have been designated the C-X-C chemokine family. Interestingly, these chemokines all exhibit between 20% and 50% homology on the amino acid level. Over the last decade, a number of C-X-C chemokines have been identified, including ENA-78.^{32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57} The second family of chemokines has the first two cysteines in juxtaposition and is called the C-C chemokine family, including MIP-1 and JE/MCP-1.^{26 27 32 33 34 41 58 59 60 61} In general, members of the C-C chemokine family have potent chemotactic activity for monocytes and lymphocytes. Our results not only suggest the importance of these chemotactic cytokines in the inflammatory response related to venous thrombosis but also highlight the importance of TNF as an early-response cytokine involved in this generation, emphasizing the importance of cytokine cascades in the pathogenesis of the inflammatory response.

CD18, a ß₂ integrin complexed to CD11b, is expressed with leukocyte activation.⁶² The cell surface expression is mediated by a variety of factors, including thrombin and C-C and C-X-C chemokines.^{29 63} Stable initial neutrophil binding followed by extravasation requires the sustained upregulation of leukocyte-dependent ß₂ integrin expression and interaction with receptor-ligand molecules on endothelial cells, such as ICAM-1.⁶⁴ TNF is an important mediator that causes the expression of ICAM-1 on the surface of endothelial cells and accentuates the adhesion of neutrophils, monocytes, and lymphocytes.^{29 65 66 67 68} Because the induction of ICAM-1 expression is predominantly regulated at the transcriptional level by IL-1 or TNF, the production of early-response cytokines, such as TNF, may play an important role for the subsequent localization/homing and recruitment of specific leukocyte populations to sites of inflammation via the induction of adhesion molecules. Our results with neutralizing ICAM-1 antibodies would support the notion that leukocyte–endothelial cell interaction in the context of thrombosis is an important event for leukocyte localization within the vein wall.

Treatment with antibody to ICAM-1 did result in an increase in neutrophils in the vein wall on day 1, as did treatment with antibody to CD18. It is possible that this effect is due to the use of a whole, intact antibody rather than only the Fab fragment of these two antibodies, with the Fc portion of the antibody binding and activating the Fc receptors on neutrophils, allowing an increase in the vein wall of these cells. Although this explanation is possible, such has not been found to be the case in other work.²⁵ Another explanation involves an ICAM-1– and CD18–independent mechanism for neutrophil extravasation, such as a P-selectin–dependent event in this model of stasis-induced venous thrombosis, given the importance of platelet aggregates in venous thrombosis³⁰ and the known upregulation of P-selectin on platelets and endothelial cells after initial activation by such agents as thrombin.^{69 70} However, although neutrophil extravasation was increased in those animals treated with antibody to ICAM-1, this antibody clearly decreased the monocyte extravasation into the vein wall later at day 6, suggesting its therapeutic efficacy.

Antibody to TNF, ICAM-1, and CD18 did result in an increase in lymphocytes in the vein wall on day 6 (Table 3). Of interest, at day 6 in the sham-dissected veins without ligation, an increase in lymphocytes to the same general range was also seen (Table 1). The reason for this lymphocyte increase is not known or addressed in the present study. However, it could relate to a ß₂- or ICAM-1–independent mechanism for lymphocyte extravasation not inhibited by antibody to TNF, ICAM-1, and CD18 or may relate in some manner to the surgical manipulation in addition to the antibodies used. It is interesting that those antibodies with the best inhibition of monocyte/macrophage extravasation (anti–ICAM-1, anti-TNF) demonstrated the greatest lymphocyte increase. Elucidation of the reason for this lymphocyte increase will become important in the understanding of the long-term inflammatory response related to venous thrombosis.

In conclusion, this study demonstrates a significant inflammatory response to venous thrombosis. This inflammation was associated with an early neutrophil infiltration into the vein wall, followed by extravasation of monocytes/macrophages and lymphocytes. Antibodies to TNF, chemokines (ENA-78, MIP-1, JE/MCP-1), and adhesion molecules (ICAM-1, CD18) partially attenuated this response. Ultimately, a decrease in the vein wall inflammatory response may result in a decline in the manifestations of chronic venous insufficiency, a national health problem.

	Acknowledgments

 
This work was supported by the University of Michigan Department of Surgery Research Advisory Committee (Dr Wakefield), NIH grants 1P50HL-46487 and HL50057 (Dr Strieter), and NIAID grants AI23521 and AI19031 (Donald C. Anderson, MD). We wish to thank Gerald D. Abrams, MD, Department of Pathology, University of Michigan, for assistance with morphometric analysis of the vein wall. We also thank Barbara Leon, Joe Martin, Addison Steele, and Donald C. Anderson for production and purification of monoclonal antibodies to ICAM-1 and CD18 (Upjohn Corp).

Received August 9, 1994; accepted November 15, 1994.

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