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Originally published online as doi:10.1189/jlb.1205718 on July 3, 2006

Published online before print July 3, 2006
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(Journal of Leukocyte Biology. 2006;80:247-257.)
© 2006 by Society for Leukocyte Biology

Endothelial growth factors VEGF and bFGF differentially enhance monocyte and neutrophil recruitment to inflammation

Sandra I. Zittermann* and Andrew C. Issekutz*,{dagger},{ddagger},1

* Departments of Pediatrics,
{dagger} Pathology, and
{ddagger} Microbiology-Immunology, Dalhousie University, Halifax, Nova Scotia, Canada

1Correspondence: Dalhousie University, Department of Pediatrics, Room 8504, 8 East Research Labs, IWK Health Centre, 5850/5980 University Avenue, Halifax, NS, Canada B3K 6R8. E-mail: Andrew.Issekutz{at}iwk.nshealth.ca

ABSTRACT

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are produced at sites of inflammation. Previously, we demonstrated that bFGF enhances leukocyte recruitment and endothelial cell adhesion molecule (CAM) expression during inflammation. Here, we investigated the influence of VEGF during acute inflammation and whether VEGF and bFGF cooperate to modulate leukocyte recruitment. Inflammation was induced in skin of rats by intradermal injection of inflammatory stimuli ± VEGF ± bFGF. Migration of 51Cr-monocytes and 111In-polymorphonuclear leukocytes (PMN) to the dermal lesions and 125I-anti-CAM monoclonal antibody binding to the dermal vasculature were quantitated after 2 h. VEGF significantly enhanced tumor necrosis factor {alpha} (TNF-{alpha})-induced monocyte recruitment by 39 ± 16% and increased P-selectin, E-selectin, and intercellular CAM-1 expression by two- to threefold over TNF-{alpha} alone. However, recruitment of monocytes to TNF-{alpha} + interferon-{gamma} (IFN-{gamma}) and of PMN to all stimuli tested was not affected by VEGF. In contrast, bFGF enhanced recruitment of both leukocyte types to all stimuli tested. With the potent TNF-{alpha} + IFN-{gamma} stimulus, in contrast to bFGF, VEGF did not enhance E-selectin or ICAM-1 expression. bFGF, but not VEGF, increased the chemotactic activity for PMN in TNF-{alpha} + IFN-{gamma}-inflamed sites by 54%. The limited effect of VEGF on these mechanisms likely contributed to the differential effect of VEGF and bFGF on leukocyte recruitment. However, VEGF + bFGF increased PMN recruitment more than did either factor alone. Thus, bFGF and VEGF differentially but synergistically enhance leukocyte recruitment to inflammatory stimuli and individually as well as jointly function as positive regulators of inflammatory cell recruitment.

Key Words: adhesion molecule • chemokine • inflammatory mediator

INTRODUCTION

Recruitment of leukocytes from the blood into tissues is a critical process in inflammation and immune responses. Leukocyte extravasation involves interaction with the vascular endothelium and response to tissue and/or vessel wall-derived molecules. Cytokines, such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin-1 (IL-1), and interferon-{gamma} (IFN-{gamma}) activate endothelial cells (EC) to express cell adhesion molecules (CAMs) recognized by leukocyte CAMs and to secrete chemokines, growth factors, and a variety of other products, which have a role in the inflammatory process [1 ]. Regulation of these EC responses by several growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF or FGF-2), and transforming growth factor-ß, has been reported [2 3 4 ].

VEGF and bFGF are potent, angiogenic factors whose activities include endothelial cell survival, proliferation, migration, and tube formation [5 ]. These growth factors are produced frequently by tumors, where they contribute to angiogenesis and tumor growth [6 ]. However, VEGF and bFGF are present as well in normal tissue and are up-regulated in chronic inflammatory reactions [7 8 9 ].

VEGF belongs to a family of homodimeric proteins of which VEGF-A is the most common member. VEGF is produced by endothelial cells, macrophages, activated T cells, and a variety of other cell types [5 ]. It was originally identified as vascular permeability factor, because of its potent action to increase endothelial permeability in vivo [10 ]. Several mechanisms have been shown to regulate VEGF expression. Among these, oxygen tension plays a major role [11 , 12 ]. However, several cytokines and growth factors also up-regulate VEGF mRNA expression and/or induce release of VEGF protein [13 , 14 ]. bFGF is a member of a family of heparin-binding growth factors. It is found in normal tissue associated with heparan sulfate on the cell surface or extracellular matrix (ECM). At sites of inflammation, bFGF is released by the action of proteases and heparinases [15 ].

VEGF and bFGF bind to tyrosine kinase receptors. Their effects are usually associated with the angiogenic activity of these growth factors. However, several reports indicate that VEGF and bFGF affect the interaction of leukocytes with the vessel wall [16 17 18 ]. In vitro models with endothelial cells as well as in vivo studies of tumors suggest an inhibitory effect of bFGF and VEGF on rolling and adhesion of leukocytes and inhibition of CAM expression on endothelium [4 , 19 ]. Conversely, in models of chronic inflammation and in chronic inflammatory diseases, where bFGF and VEGF are abundant in the involved tissue, a proinflammatory role has been attributed to these growth factors [7 , 20 , 21 ]. Previously, our laboratory demonstrated in vivo that bFGF enhances the recruitment of monocytes, T cells, and polymorphonuclear leukocytes (PMN) to inflamed dermal sites. One of the mechanisms involved in this enhancement was found to be up-regulation of CAM expression in the inflamed tissue in response to EC-activating cytokines [22 ]. However, as bFGF can induce VEGF in some cells [23 ], the contribution of VEGF to these effects of bFGF warrants further study.

Because of the reported inhibitory effect of VEGF on EC activation and leukocyte recruitment in the tumor-associated studies on the one hand and its potential proinflammatory effect in chronic inflammatory diseases on the other, the effect of VEGF during inflammation requires clarification. Therefore, we used a model of acute dermal inflammation in which timing and stimuli are controlled to assess the influence of VEGF on monocyte and PMN recruitment and vascular permeability. We also examined whether VEGF and bFGF may have cooperative actions on acute inflammation and the mechanisms involved.

MATERIALS AND METHODS

Animals
Inbred male Lewis rats (Charles River Canada, St-Constant, Quebec, Canada), weighing 250–300 g, were used. Some animals were used exclusively for harvesting of blood leukocytes for purification, as described previously [24 ], and others were used for dermal inflammatory reactions and were recipients of labeled leukocytes.

Reagents
Human VEGF (38.2 kD), human bFGF, rat TNF-{alpha}, and rat IFN-{gamma} were purchased from PeproTech Inc. (Rocky Hill, NJ). Heparin (molecular weight, approximately 3000 Da, from porcine intestinal mucosa) was from Sigma-Aldrich Canada (Oakville, Ontario). Human serum albumin (HSA), endotoxin- and pyrogen-free, was from Canadian Blood Services (Ottawa, Ontario). RPMI-1640 (Sigma-Aldrich Canada) and all salts for buffers [Tyrode’s solution (TyS), phosphate-buffered saline (PBS)] were made with analytical-grade chemicals and dissolved in endotoxin- and pyrogen-free water (Abbott Laboratories, Saint Laurent, Quebec, Canada). The following anti-CAM mouse immunoglobulin G (IgG) monoclonal antibodies (mAb) were used: RMP-1 (to rat P-selectin) [25 ], RME-1 (to rat E-selectin) [26 ], 5F10 {to rat vascular CAM-1 (VCAM-1); a kind gift from Linda Burkley, Biogen Inc., Cambridge, MA [27 ]}, and 1A29, WT-3, and TA-2 {to rat intercellular CAM-1 (ICAM-1), rat CD18, and rat very late antigen-4 (VLA-4), respectively; kind gifts from Masayuki Miyasaka, Research Institute for Microbial Disease, Osaka, Japan, and Thomas B. Issekutz, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada [28 29 30 ]}.

Rat leukocyte purification and labeling
Rat blood PMN and monocytes for migration studies were isolated by hydroxyethyl starch exchange transfusion technique as described previously [24 ]. Briefly, via the femoral vein of an anesthetized, heparinized donor rat, blood was gradually exchanged with 6% hydroxyethylstarch/saline (Hespan; American Hospital Supply Corp., McGaw Park, IL) into acid-citrate-dextrose (ACD; formula A, Fenwal-Travenol, Malton, Ontario, Canada), and red blood cells were sedimented at 1 g. The leukocyte-rich plasma was harvested, and leukocytes were pelleted (200 g for 10 min) and resuspended in Ca++Mg++-free TyS (TyS–/–) containing 10% platelet poor plasma (PPP). Leukocytes were layered on discontinuous plasma (10%)-TyS–/–-Percoll gradients (63%/74%). Cells recovered at the interface between 63% and 74% Percoll were >95% neutrophils. The PMN were washed and labeled with 111In-labeled oxine (Amersham Corp., Oakville, Ontario, Canada) at 1.5 µCi/1 x 107 PMN in 0.1 ml TyS–/– for 10 min at room temperature. Labeled PMN were washed three times with and resuspended in TyS–/– 10% PPP for intravenous (i.v.) injection.

Monocytes were purified as described previously [24 ] with minor modifications. Briefly, the mononuclear layer on the top of 63% Percoll was removed, washed, and resuspended in 3.6 ml TyS–/– 10% PPP, and the osmolarity of this suspension was increased slightly by the addition of 20 µl 9% NaCl to improve the separation of monocytes from lymphocytes. After incubation for 10 min at 37°C, this cell suspension was layered onto another discontinuous plasma-TyS–/– Percoll gradient composed of 40% Percoll, on top of 55% Percoll, on top of 59% Percoll. All of the Percoll layers had 12.5 µl 9% NaCl/2.5 ml gradient, which was centrifuged at 400 g for 30 min at room temperature, and monocytes were harvested at the 40/55% and the 55/59% interfaces. The purified monocytes (>85%) were washed and radiolabeled with 75 µCi Na251CrO4 (Amersham Corp.) per 5 x 107 monocytes in 1 ml TyS–/–-10% PPP for 30 min at 37°C. Labeled monocytes were washed twice and resuspended in TyS–/–-10% PPP for i.v. injection. Each rat received 5 x 106 51Cr-labeled monocytes together with 10 x 106 111In-labeled PMN.

Vascular permeability and blood flow measurements
Exudation as a result of enhanced vascular permeability was quantitated using HSA labeled with 125I, as described previously [31 ]. Briefly, 5 µCi 125I-HSA/kg was administered i.v. 2 h before sacrifice. The content of 125I per site and of 125I per µl blood plasma was quantified, and content in skin sites is expressed as µl plasma albumin equivalents/site.

Blood flow was measured as described previously [32 ] with minor modifications. Briefly, just before sacrifice, 50 µCi 86RbCl (Amersham Corp.) was injected i.v. Forty-five seconds later, 1 ml saturated KCl solution was injected i.v. to cause cardiac arrest. The content of 86Rb in the skin sites was quantified and expressed as fold increase over the control sites.

Growth factor treatments and inflammatory reactions
The dorsal skin of Lewis rats was shaved, and dermal sites were injected intradermally (i.d.) in triplicate with 50 µl 50 ng VEGF or 50 ng bFGF or VEGF + bFGF in diluent (RPMI 1640-0.1% HSA-5 µg heparin) in the absence of or together with inflammatory stimuli. As VEGF or bFGF with heparin did not induce leukocyte recruitment, for the purposes here, these were not considered "inflammatory stimuli", which included 3 ng TNF-{alpha}, 100 U IFN-{gamma}, and 3 ng TNF-{alpha} + 100 U IFN-{gamma}, all in diluent. Inflammatory agents were injected i.d. at the time of i.v. injection of radiolabeled leukocytes. The animals were killed 2 h later.

In some experiments, the recruitment of leukocytes to inflamed sites was blocked by i.v. injection of anti-rat CD18 + anti-rat CD49d antibodies (WT-3, 1 mg/rat, and TA-2, 0.5 mg/rat) immediately before induction of inflammation and injection of radiolabeled leukocytes. The absence of labeled leukocyte accumulation after 2 h was verified as reported previously [33 34 35 ].

Measurement of leukocyte accumulation
At the time of sacrifice, 2 ml blood was collected in ACD anticoagulant, and dorsal skin was removed and cleaned. The inflamed sites were punched out (12-mm diameter punch). Samples of spleen, liver, lung, and lymph nodes were taken for determination of 111In and 51Cr content. Counts of 111In and 51Cr in the tissues are expressed as counts per minute (cpm) per 106 cpm injected. The amount of radioactivity (111In, 51Cr, 125I) was measured in a four-channel {gamma}-spectrometer (LKB1282, Fisher Scientific, Dartmouth, NS). Automatic corrections were made for the spill of isotope emissions into adjacent channels.

Radiolabeling of mAb
The in vivo expression on endothelial cells of E-selectin, P-selectin, ICAM-1, and VCAM-1 was quantified by the binding in the skin sites of i.v.-administered 125I or 131I-labeled antibodies as described previously [36 ]. Briefly, 100 µg mAb was incubated with 100 µCi 125I or 131I in an Iodo-Gen precoated tube (MJS BioLynx Inc., Brockville, Ontario, Canada) for 15 min at room temperature. Removing the sample from the tube stopped the reaction. The free isotope was separated by overnight dialysis against PBS and then diluting and reconcentrating the sample in a centrifugal filter device with a 30,000 nominal molecular weight cut-off limit (Millipore Corp., Bedford, MA) until 99% of the 125I was mAb-associated.

Quantification of vascular adhesion molecule expression with radiolabeled mAb
Endothelial expression of E-selectin, P-selectin, ICAM-1, and VCAM-1 in dermal-inflamed sites was determined by i.v. injection of 125I-labeled RME-1 (anti-rat E-selectin), RMP-1 (anti-rat P-selectin), 1A29 (anti-rat ICAM-1), or 5F10 (anti-rat VCAM-1) mAb plus 131I-labeled isotype control IgG [mAb 11D10 IgG2a anti-human neutrophil (unpublished) not reactive with rat cells or mAb 3H11 IgG1 anti-pertussis toxin]. Briefly, the animals were anesthetized with Ketamine-Innovar subcutaneously (s.c.), and 10 min later, a mix of 25 µg 125I anti-CAM mAb plus 25 µg isotype control IgG was injected i.v. Immediately thereafter, the animals received an intraperitoneal injection of 0.4 ml xylazine. Exactly 3 min later, the abdomen was opened, the ascending aorta was cannulated, 1 ml 200 U heparin in saline was administered via this cannula, and the inferior vena cava was opened, and the animals were perfused via the aorta with 75 ml TyS+/+ buffer. Skin sites were then removed, and the content of 125I and 131I in the sites was quantified in a {gamma}-counter along with a known fraction of the total injected dose (ID) of each labeled antibody. The expression of CAMs in test and control-injected skin sites was calculated as percent of the 125I-anti-CAM ID – percent 131I ID of isotype control IgG.

Inflamed tissue fluid retrieval
Acute air pouches (two per animal) were generated by s.c. injection on the back of 1.5 ml sterile air + 25 ng TNF-{alpha} ± 1200 U IFN-{gamma} ± 50 ng VEGF or ± 50 ng bFGF in 0.5 ml diluent. After 1 h, the animals were anesthetized with 0.4 ml Ketamine-Innovar s.c., and the pouches were opened. The connective tissue was recovered, minced at 4°C, and centrifuged at 10,000 g. The supernatants were collected and frozen at –80°C.

Measurement of chemotactic activity of inflammatory exudates
PMN were purified and labeled with 111In-oxine as described above. Air-pouch fluid samples were diluted (1/10 or 1/30) in RPMI-0.5% HSA-10 mM HEPES, and 30 µl was seeded in triplicate in a Chemo TX System plate (NeuroProbe Inc., Gaithersburg, MD). For PMN chemotaxis, a 3-µm membrane was applied over the wells, and 25 x 103 111In-labeled PMN in 60 µl RPMI-0.5% HSA-10 mM HEPES were seeded on the top and incubated at 37°C. After 45 min, the membrane was removed, the undersurface swabbed to remove transmigrated cells, and the swab and the cells in the lower chamber were recovered and counted in a {gamma}-counter.

Statistical analysis
Statistical significance was determined using paired t-tests when two conditions were compared and ANOVA with Bonferroni for multiple comparisons test when more than two conditions were compared. P values exceeding 0.05 were considered not significant.

RESULTS

VEGF enhances the recruitment of leukocytes in response to inflammation
To investigate the effect of VEGF on acute inflammation and leukocyte recruitment, dorsal skin of rats was injected (i.d.) with inflammatory cytokines (TNF-{alpha}, IFN-{gamma}, TNF-{alpha}+IFN-{gamma}), individually or together with 50 ng VEGF in diluent (5 µg heparin in RPMI-HSA). For comparison, bFGF (50 ng in same diluent) was used with the same agents. Heparin was used as a cofactor, as it is known to bind to VEGF (and bFGF), and provided more consistent results with the growth factors (not shown). Heparin was used at the lowest concentration (5 µg), which avoided any effect on local hemostasis. The doses of inflammatory stimuli were selected to induce submaximal leukocyte accumulation (~50% maximal for each stimulus), allowing the detection of any modulation (increase or inhibition) in the recruitment of leukocytes. Neither bFGF nor VEGF induced recruitment of radiolabeled monocytes or PMN to dermal sites in the absence of the inflammatory stimuli (see Diluent bars in Figs. 1 and 2 ). However, VEGF enhanced recruitment of monocytes in response to TNF-{alpha} or IFN-{gamma} (139±16% and 183±29% of control, respectively, P<0.05). This effect was similar to bFGF. Despite this enhancing effect of VEGF with TNF-{alpha} alone or IFN-{gamma}, VEGF neither increased nor decreased recruitment with the most potent stimuli, i.e., the combination of TNF-{alpha} with IFN-{gamma}. In marked contrast, bFGF potentiated monocyte recruitment under all conditions tested (between 155±21% and 212±14% of control, P<0.05).


Figure 1
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  		Figure 1. Effect of VEGF and bFGF on monocyte recruitment to inflammatory stimuli. Dorsal skin of Lewis rats was injected i.d. with diluent (RPMI-HSA-heparin) or 50 ng VEGF + diluent or 50 ng bFGF + diluent, with or without inflammatory stimuli (3 ng TNF-{alpha}, 100 U IFN-{gamma}, 3 ng TNF-{alpha}+100 U IFN-{gamma}). Immediately after, 51Cr-labeled monocytes were injected i.v. After 2 h, the skin was removed, and 51Cr in the dermal sites was quantitated. Values are means ± SEM percent of control (Diluent) sites, which are set as the 100% response. The mean absolute 51Cr-monocyte accumulation per lesion is given under each bar pair (n=4–11). *, P < 0.05; ***, P < 0.005, paired t-test.

 

Figure 2
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  		Figure 2. Effect of VEGF and bFGF on PMN recruitment to inflammatory stimuli. Skin sites were treated as in Figure 1 , and then 111In-labeled PMN were injected i.v. After 2 h, 111In in the sites was quantitated. Values are mean ± SEM percent of control sites expressed as in Figure 1 (n=4–14). *, P < 0.05, paired t-test.

 
The recruitment of PMN in response to any of the stimuli tested was not affected by VEGF (Fig. 2) . In contrast, bFGF significantly enhanced the recruitment of PMN in response to TNF-{alpha} + IFN-{gamma} and TNF-{alpha} (195±22 and 232±18, respectively, P<0.05). PMN migration to IFN-{gamma}-injected sites was minimal, as reported before [24 ], and therefore, is not shown in Figure 2 .

The VEGF and bFGF doses selected were based on dose-response experiments between 10 and 200 ng VEGF and 2 and 200 ng bFGF. The maximum responses for bFGF and VEGF were observed with 50 ng. The different doses of VEGF used (10–200 ng) did not affect monocyte or PMN recruitment to TNF-{alpha} + IFN-{gamma}-induced inflammation (not shown).

The enhanced monocyte recruitment induced by VEGF does not correlate with increased vascular permeability
We investigated whether the enhancement of monocyte recruitment by VEGF was related to any increase in vascular permeability induced by VEGF. As is shown in Figure 3 , VEGF in diluent alone increased the plasma albumin exudation by 186% of control sites. Furthermore, VEGF increased the vascular permeability in TNF-{alpha} (286±57% of control)-, IFN-{gamma} (175±21% of control)-, and TNF-{alpha} + IFN-{gamma} (236±15% of control)-inflamed sites (P<0.05). VEGF enhanced plasma albumin exudation to similar levels (13–14 µl plasma/lesion) in the absence of inflammatory stimuli, as it did in IFN-{gamma}-inflamed sites. However, recruitment of monocytes in the absence of additional inflammatory stimuli was not induced by VEGF alone, but it was increased by VEGF in response to IFN-{gamma}. Furthermore, in TNF-{alpha} + IFN-{gamma}-inflamed sites, VEGF increased vascular permeability but did not affect PMN or monocyte recruitment to this stimulus (Figs. 1 2 3) .


Figure 3
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  		Figure 3. Effect of treatment of dermal sites with VEGF or bFGF on vascular permeability. Skin sites were pretreated as in Figure 1 , followed by i.v. injection of 5 µCi/kg 125I-HSA. After 2 h, 125I in the sites was quantitated. Values are means ± SEM percent of control sites expressed as in Figure 1 (n=4). *, P < 0.05; **, P < 0.01; ***, P < 0.005, paired t-test.

 
In contrast, bFGF did not induce an increase in vascular permeability under any of the conditions tested (Fig. 3) , yet it significantly increased the recruitment of monocytes and PMN (Figs. 1 and 2) .

Inflammatory hyperemia is known to enhance PMN recruitment induced by recruiting mediators [32 ]. Therefore, the blood flow was quantified with 86RbCl uptake. Blood flow (expressed as fold increase over the control diluent-injected sites) was enhanced in TNF-{alpha} + IFN-{gamma} induced inflammation (1.7±0.5, P<0.05) but not by IFN-{gamma} alone. However, VEGF or bFGF alone or in combination with these stimuli did not alter the blood flow in the sites (data not shown).

VEGF and bFGF in combination enhance monocyte and PMN recruitment to inflammatory cytokines
As VEGF and bFGF signal through different tyrosine-kinase receptors and are also coexpressed frequently in inflammatory diseases, we investigated their effect when combined. The combination of bFGF + VEGF did not induce PMN recruitment in the absence of additional inflammatory stimuli (diluent: 291±48 vs. VEGF+bFGF: 335±46 cpm/site). Figure 4a shows that cotreatment of dermal sites with bFGF + VEGF additively increased the recruitment of monocytes in response to TNF-{alpha}, compared with TNF-{alpha} + VEGF or TNF-{alpha} + bFGF (136±12% and 153±18% of control, respectively; P<0.01). As described above, VEGF did not have any effect on PMN recruitment to TNF-{alpha}-induced inflammation. However, the cotreatment of dermal sites with bFGF + VEGF significantly increased the recruitment of PMN to TNF-{alpha} when compared with bFGF alone (266±38% vs. 232±35% of control; P<0.05; Fig. 4b ). Monocyte recruitment in response to the stronger stimuli, such as TNF-{alpha} + IFN-{gamma}, was enhanced by the combination of bFGF + VEGF to a similar level as induced by bFGF (Fig. 4c) . A similar effect of bFGF + VEGF was observed on the recruitment of PMN in response to TNF-{alpha} + IFN-{gamma} as with only bFGF (VEGF+bFGF: 260±12% vs. bFGF: 221±10% of control; P<0.05; Fig. 4d ).


Figure 4
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  		Figure 4. Effect of cotreatment of dermal sites with VEGF + bFGF on recruitment of monocytes and PMN to inflammatory cytokines. Dermal sites were treated with inflammatory stimulus (3 ng TNF-{alpha} or 3 ng TNF-{alpha}+100 U IFN-{gamma}, as indicated) ± VEGF ± bFGF. Immediately after, 51Cr-monocytes (a and c) or 111In-PMN (b and d) were administered i.v. After 2 h, the 51Cr and 111In in the dermal sites were quantitated. Values are mean ± SEM percent of control sites (n=8–16). *, P < 0.05; **, P < 0.01; ***, P < 0.005, ANOVA with Bonferroni for multiple comparisons.

 
We compared vascular permeability in bFGF + VEGF-treated sites with VEGF alone in TNF-{alpha} + IFN-{gamma}-induced inflammation as a possible mechanism for the enhancement of monocyte or PMN recruitment. We observed that the combination of growth factors did not enhance vascular permeability beyond the effect of VEGF alone in TNF-{alpha} + IFN-{gamma}-inflamed sites (bFGF+VEGF: 20±1 vs. VEGF: 22±1 µl plasma/site; n=4).

VEGF enhances CAM expression induced by TNF-{alpha} but not by TNF-{alpha} + IFN-{gamma}
We investigated the modulation of CAM expression as one possible mechanism by which VEGF might affect leukocyte recruitment. We observed that in dermal sites treated with VEGF + diluent, the expression of P-selectin was enhanced significantly compared with diluent alone [VEGF: 4±1% vs. diluent: 2±1% of ID ({Delta}ID)x10–3; P<0.05], but there was no effect of VEGF alone on E-selectin or ICAM-1 expression (data not shown). Injection of bFGF + diluent induced a significant increase in the expression of ICAM-1 and P-selectin in the absence of inflammatory cytokines (ICAM-1: 4.6±0.6% and P-selectin: 3.3±0.8%). Furthermore, VEGF enhanced P-selectin expression in TNF-{alpha}-stimulated sites to a similar level as bFGF (TNF-{alpha}+VEGF: 17±3% vs. TNF-{alpha}+diluent: 10±2%; P<0.01; Fig. 5a ). A similar effect was observed on the expression of E-selectin and ICAM-1 during TNF-{alpha} stimulation (Fig. 5b and 5c , respectively). Although bFGF but not VEGF enhanced PMN recruitment to TNF-{alpha}, the enhancement of E- and P-selectin and ICAM-1 expression induced by both growth factors was comparable. Moreover, the combination of bFGF + VEGF in TNF-{alpha}-stimulated sites did not have an additive or synergistic effect on the expression of CAMs, when compared with each growth factor individually. VCAM-1 expression was not observed in nonstimulated sites, and TNF-{alpha}, VEGF, or bFGF did not induce detectable expression (data not shown).


Figure 5
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  		Figure 5. Effect of VEGF and bFGF on adhesion molecule expression in TNF-{alpha}-inflamed sites. The dorsal skin of rats was injected i.d. with 3 ng TNF-{alpha} ± 50 ng VEGF ± 50 ng bFGF in diluent. After 2 h, 25 µg 125I-labeled anti-P-selectin mAb (RMP-1; a), anti-E-selectin mAb (RME-1; b), or anti-ICAM-1 mAb (1A29; c) + 25 µg 131I-labeled isotype control IgG were injected i.v. and allowed to circulate for 3 min. The animals were then killed, the skin sites removed, and 125I and 131I quantitated. CAM expression is expressed as the mean ± SEM of the specific anti-CAM mAb bound in the site [% ID of anti-CAM mAb–% ID of isotype control mAb (131I); n=4–6]. *, P < 0.05; **, P < 0.01, ANOVA with Bonferroni for multiple comparisons.

 
In TNF-{alpha} + IFN-{gamma}-stimulated sites, CAM expression was two- to fivefold higher than for TNF-{alpha} alone (Figs. 5 and 6 ). As is shown in Figure 6 , bFGF significantly, further enhanced the expression of E-selectin (E-selectin: bFGF 13±0.75 vs. diluent 8±0.5; P<0.01) and ICAM-1 (ICAM-1: bFGF 14±0.8 vs. diluent 7±0.9; P<0.01). In contrast, VEGF enhanced the expression of only P-selectin in TNF-{alpha} + IFN-{gamma}-treated sites (30±3.3; P<0.05; Fig. 6a ) without any effect on E-selectin and ICAM-1 expression. The combination of bFGF + VEGF enhanced P-selectin expression to a similar level, as did each growth factor individually (32±4.7; P<0.05) and E-selectin expression to similar levels as bFGF (12±1.1; P<0.05; Fig. 6a and 6b ). In contrast, not only did VEGF not increase ICAM-1 expression when it was combined with bFGF in TNF-{alpha} + IFN-{gamma}-inflamed tissue but rather, it inhibited the up-regulation when compared with bFGF alone (TNF-{alpha}+IFN-{gamma}+bFGF: 14±0.8; TNF-{alpha}+IFN-{gamma}+bFGF+VEGF: 11±0.7; P<0.01; Fig. 6c ). VCAM-1 expression was induced by TNF-{alpha} + INF-{gamma} stimulation (2.8±1; P<0.05, n=4); however, bFGF, VEGF, or the combination of both growth factors did not enhance the expression further.


Figure 6
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  		Figure 6. Effect of VEGF and bFGF on adhesion molecule expression in TNF-{alpha} + IFN-{gamma}-inflamed sites. The dorsal skin of rats was injected i.d. with 3 ng TNF-{alpha} + 100 U IFN-{gamma} ± 50 ng VEGF ± 50 ng bFGF in diluent. Thereafter, the procedure was as in Figure 5 [(a) P-selectin; (b) E-selectin; (c) ICAM-1]. CAM expression is expressed as the mean ± SEM of the specific anti-CAM mAb bound in the site [% ID of anti-CAM mAb–% ID of isotype control mAb (131I); n=4–8]. *, P < 0.05; ***, P < 0.01, ANOVA with Bonferroni for multiple comparisons.

 
We investigated whether the increase of CAM expression induced by growth factors could be an indirect effect secondary to an increase in the infiltrating leukocytes. Therefore, monocyte and PMN recruitment was blocked by i.v. injection of anti-CD18 + anti-CD49d mAb, and the expression of E- and P-selectin induced by TNF-{alpha} + IFN-{gamma} ± VEGF ± bFGF was evaluated. As is shown in Table 1 , the complete blockade of PMN migration to the inflamed sites slightly reduced the expression of E- and P-selectin compared with the normal animal. However, the enhancement of these CAMs induced by bFGF ± VEGF was still observed. Previous studies have shown that this mAb treatment completely blocks monocyte and lymphocyte recruitment as well [34 , 37 ].


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  		Table 1. Effect of Blockade of PMN Recruitment on Up-Regulation of P- and E-Selectin Expression by bFGF, VEGF, and VEGF + bFGF in TNF-{alpha} + IFN-{gamma}-Inflamed Dermal Sites

 
bFGF but not VEGF increases PMN chemotactic activity in inflamed tissue fluid
We showed above that VEGF, in contrast to bFGF, did not increase the recruitment of monocytes and PMN in response to TNF-{alpha} + IFN-{gamma}. Furthermore, the up-regulation of CAMs by these growth factors did not correlate with their differential effects on monocyte and PMN recruitment. These results suggested that another mechanism might be involved in the enhancement, especially of PMN recruitment by bFGF. Therefore, we investigated the chemotactic activity for PMN in inflamed tissue fluid generated by s.c. injection of TNF-{alpha} + IFN-{gamma} ± bFGF ± VEGF. Fluid retrieved from TNF-{alpha} + IFN-{gamma}-inflamed tissue induced significant chemotaxis of PMN (233% of diluent control; P<0.05). Furthermore, bFGF enhanced the chemotactic activity in TNF-{alpha} + IFN-{gamma}-retrieved tissue fluid by 54% (P<0.01) within 1 h of induction of inflammation. In contrast, VEGF had no such effect on the PMN chemotactic activity in the tissue fluid from TNF-{alpha} + IFN-{gamma} sites (Fig. 7 ).


Figure 7
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  		Figure 7. Effect of VEGF and bFGF on chemotactic activity for PMN. Rats were injected s.c. with 1.5 ml sterile air + 0.5 ml diluent ± 25 ng TNF-{alpha} + 1200 U IFN-{gamma} ± 50 ng VEGF or 50 ng bFGF. Inflamed tissue fluid was collected after 1 h, and its activity for inducing migration of 111In-PMN was evaluated. The PMN chemotaxis is expressed as mean ± SEM percent of diluent-injected sites (n=5–9). **, P < 0.01, paired t-test.

 
DISCUSSION

VEGF and bFGF are critical mediators of physiological as well as pathological angiogenesis. During tissue injury, there is production and/or release of bFGF and VEGF, and increased levels have been reported in several chronic, inflammatory diseases. However, their role in inflammation is not clear. We recently demonstrated that bFGF increases the recruitment of monocytes, T cells, and PMN to inflammatory dermal sites [22 ]. Here, we extend these in vivo studies to define the influence of VEGF and the concurrent effects of VEGF and bFGF on the vascular responses and leukocyte recruitment during acute inflammation.

VEGF is reported to be chemotactic for monocytes in vitro [38 ]. However, we did not observe any effect of VEGF or bFGF alone on acute monocyte or PMN recruitment in vivo. This difference likely reflects the complexity of interactions of VEGF in vivo with other tissue cells and ECM. However, VEGF significantly enhanced recruitment of monocytes to dermal sites stimulated with TNF-{alpha} or IFN-{gamma}. This result is consistent with previous reports showing that VEGF expression in a skin allograft model correlated with increased leukocyte recruitment to the graft [20 ] and that an anti-VEGF antibody significantly inhibited CD68+ monocyte/macrophage recruitment to the graft. Similarly, Sauder and co-workers [39 ] demonstrated that VEGF increased in a contact-hypersensitivity reaction, and an anti-VEGF mAb decreased the response by reducing dermal cellular infiltrates. Here, we extend those findings by showing that in the acute inflammatory response (2 h), VEGF has a synergistic action with TNF-{alpha} or IFN-{gamma} for the recruitment of monocytes. However, our results suggest that when stronger stimuli are involved, such as the synergistic, proinflammatory combination of TNF-{alpha} + IFN-{gamma}, VEGF no longer modulates the recruitment of monocytes or PMN (Fig. 1) . In contrast, bFGF strongly enhanced monocyte and PMN recruitment to all inflammatory cytokines tested by an acute and synergistic effect. Our study, with respect to various stimuli, extends the in vivo findings of Yamashita et al. [40 ] in rat adjuvant arthritis, indicating that bFGF overexpression in arthritic joints aggravates inflammation.

VEGF is a potent enhancer of vascular permeability [10 ], and this could account for the increased leukocyte recruitment [41 ]. As expected, VEGF treatment increased vascular permeability in all conditions tested, including when administered alone (Fig. 3) . However, only the recruitment of monocytes in response to TNF-{alpha} or IFN-{gamma} was enhanced by VEGF. PMN recruitment was not enhanced by VEGF with the inflammatory conditions tested. In contrast, bFGF did not affect vascular permeability but increased monocyte and PMN recruitment significantly in response to cytokine-induced inflammation. These results demonstrate that neither the VEGF nor the bFGF enhancement of monocyte and PMN recruitment to inflammatory stimuli is via a permeability mechanism.

The presence of bFGF and VEGF in tumors or inflammatory diseases and even the induction of VEGF by bFGF have been described [8 , 21 , 23 , 42 , 43 ]. We demonstrated here that VEGF and bFGF independently enhanced the recruitment of monocytes and/or PMN to inflammatory cytokines. When dermal sites were cotreated with VEGF + bFGF, the recruitment of monocytes to TNF-{alpha} and PMN to TNF-{alpha} and TNF-{alpha} + IFN-{gamma} was increased additively or synergistically. Even when VEGF alone did not affect PMN recruitment in response to inflammatory cytokines, it did not inhibit bFGF enhancement but rather potentiated it. Our observations show for the first time that VEGF + bFGF synergistically potentiates inflammation induced by inflammatory cytokines by enhancing the recruitment of monocytes and PMN and that this effect is stronger than with each individual growth factor.

One of the mechanisms involved in leukocyte recruitment, which could be a target of modulation by products released during inflammation, is the expression of CAMs for leukocytes by vascular endothelial cells. Several reports have described modulation of CAMs by endothelial growth factors [44 45 46 47 ]. Detmar et al. [16 ] reported enhancement of leukocyte rolling and adhesion but not of leukocyte infiltration in skin of VEGF-transgenic mice. Anti-P- and anti-E-selectin antibodies inhibited the increased rolling and adhesion of leukocytes observed. Furthermore, an anti-VCAM-1 antibody normalized the enhanced leukocyte adhesion, suggesting that up-modulation of these specific CAMs in the VEGF-overexpressing mice was responsible for the observed increase in leukocyte rolling and adhesion. These mice also had increased densities of cutaneous microvasculature, secondary to angiogenesis. Our results are independent of angiogenesis, as the VEGF treatment used in our experiments was only 2 h when PMN and monocyte recruitment is maximal during acute inflammation, as reported previously [24 ], but this time-frame is insufficient for angiogenesis. Similarly to Detmar et al. [16 ], in the absence of inflammatory mediators, we did not observe PMN and monocyte recruitment in the skin sites induced by VEGF. However, P-selectin expression was enhanced by VEGF + diluent (see Results). This indicates that up-regulation of P-selectin alone is not sufficient to induce emigration of PMN or monocytes to the tissue. Furthermore, VEGF enhanced P-selectin but not E-selectin and ICAM-1 expression induced by TNF-{alpha} + IFN-{gamma} (Fig. 6) . The inability of VEGF to enhance E-selectin and ICAM-1 expression cannot be interpreted as a plateau effect as a result of maximal stimulation by the synergistic combination of TNF-{alpha} + IFN-{gamma}, as bFGF further enhanced the expression induced by TNF-{alpha} + IFN-{gamma}. Taken together, our results suggest that in response to a strong stimulus such as TNF-{alpha} + IFN-{gamma}, the inability of VEGF to further up-regulate E-selectin and ICAM-1 expression may in part explain its failure to enhance monocyte and PMN recruitment. VEGF significantly increased the expression of E- and P-selectin and ICAM-1 when CAM up-regulation was relatively weak (TNF-{alpha} alone, compared with TNF-{alpha}+IFN-{gamma}). However, CAM up-regulation alone may not be sufficient to enhance leukocyte emigration, as additional factors, especially the generation of chemoattractants locally, are also required [1 ]. It has been demonstrated that VCAM-1-VLA-4 interactions support monocyte transendothelial migration and recruitment [48 49 50 51 ]. Our findings here indicate that during acute inflammation induced by TNF-{alpha}, VCAM-1 expression was undetectable, and bFGF or VEGF did not induce VCAM-1. However stronger stimuli, such as TNF-{alpha} + IFN-{gamma}, induced detectable levels of VCAM-1 expression, but this was not modified by the growth factors tested, suggesting that the enhancement of monocyte recruitment by bFGF and VEGF is independent of VCAM-1 modulation. Our and Detmar’s observations contrast with those of Griffioen and co-workers [4 , 18 ] that VEGF and bFGF significantly inhibit ICAM-1, VCAM-1, and E-selectin expression on cytokine-stimulated endothelial cells. However, this appears to be time-dependent, e.g., bFGF and VEGF rapidly up-regulate the expression of ICAM-1 and VCAM-1 on endothelium in vitro, but prolonged bFGF exposure inhibits the expression of ICAM-1, VCAM-1, and E-selectin on TNF-{alpha}-stimulated endothelium [19 , 52 ].

The up-regulation of CAMs by bFGF or VEGF was not a secondary effect of the infiltrating leukocytes, as when the recruitment of PMN (and monocytes or T cells, as referenced in refs. [33 , 34 ]), in response to TNF-{alpha} + IFN-{gamma}, was blocked by >98%, P-selectin and/or E-selectin expression was still increased by VEGF, bFGF, and VEGF + bFGF treatment (Table 1) . Thus, the mechanism of bFGF and VEGF potentiation of CAM expression in response to inflammatory cytokines is likely to be local at the level of the endothelium or perhaps secondary via an effect on other resident cells, which may costimulate the microvascular endothelium. It is interesting to note that the blocking of leukocyte recruitment to the inflamed sites also reduced the level of expression of P- and E-selectin when compared with nonblocked animals. This raises the possibility that the infiltrating leukocytes release soluble factors, which might contribute to enhance the CAM expression, or the interaction of leukocytes during transendothelial migration may further enhance the activation of endothelial cells induced by inflammatory mediators present.

It could be argued that the effect observed for bFGF in inflammatory reactions might be indirect through the induction of VEGF, as such induction has been observed, e.g., in smooth muscle cells [23 ]. Our results do not support this mechanism, as bFGF but not VEGF enhanced monocyte recruitment induced by TNF-{alpha} + IFN-{gamma}, and in contrast to bFGF, VEGF had no effect on PMN recruitment induced by inflammatory cytokines. Furthermore, VEGF inhibited the enhanced ICAM-1 expression induced by bFGF in TNF-{alpha} + IFN-{gamma}-induced lesions, although this modulation did not alter the enhanced PMN recruitment. Finally, bFGF did not alter vascular permeability under any of the conditions tested, and VEGF uniformly increased albumin extravasation in the dermal lesions. Thus, at least in skin, the acute effect of bFGF on the inflammatory reaction, as assessed by leukocyte recruitment, is independent of VEGF.

Modulation of chemokines has been described for bFGF and VEGF [3 , 19 , 20 , 53 ]. Reinders et al. [20 ] demonstrated that the treatment of endothelial cells with VEGF enhanced the production of monocyte chemoattractant protein-1 (MCP-1) and IL-8 and that VEGF synergized with IFN-{gamma} in the production of IFN-inducible protein 10. Similarly, increased production of MCP-1 was described by stimulation of bovine retinal microvascular endothelial cells in a concentration- and time-dependent manner [53 ]. In human brain microvascular endothelial cells, VEGF (and to a lesser degree and later in time, bFGF) induced IL-8 expression [3 ]. Our results show that TNF-{alpha} + IFN-{gamma}-inflamed tissue fluid induced PMN migration in vitro, compared with diluent control. It is interesting that bFGF but not VEGF increased this chemotactic activity. This apparent difference in the capacity of VEGF to enhance the chemotactic activity in the inflammatory fluid may in part explain why bFGF enhanced PMN recruitment to TNF-{alpha} + IFN-{gamma}, but VEGF did not. This fact, in conjunction with the absence of an enhancing effect of VEGF on E-selectin and ICAM-1 expression in the TNF-{alpha} + IFN-{gamma} lesions, again, in contrast with bFGF, likely contributes to the significantly different effects of bFGF and VEGF on PMN recruitment during intense acute inflammation, as induced by the synergistic combination of TNF-{alpha} + IFN-{gamma}. The differing observation here from previous reports regarding chemotactic factor stimulation by VEGF may be related to the time-point selected to harvest the inflammatory fluid, i.e., 1 h. This time was selected, as the chemotactic activity is expected to precede maximal PMN recruitment to the tissue, which is reached between 1 and 2 h. The chemokine induction by VEGF reported by others [3 , 20 , 53 ] was 1 h or later for mRNA expression and later time-points for protein detection.

In conclusion, we demonstrate that neither VEGF nor bFGF induces PMN or monocyte recruitment in vivo to dermal inflammation, but both potentiate cytokine-induced, endothelial-dependent PMN and monocyte recruitment. However, bFGF enhancement of recruitment is to a wider variety of inflammatory stimuli and to a greater extent for PMN and monocytes. The difference between bFGF and VEGF potentiation of monocyte recruitment seems to reside in the ability of bFGF, but not VEGF, to enhance the up-regulated expression of E-selectin and ICAM-1 by strong stimuli, such as TNF-{alpha} + IFN-{gamma}, which are often coexpressed in tissue during immunologic or infection-induced inflammation [54 ]. Furthermore, the enhancement of chemotactic activity for PMN by bFGF, but not VEGF, might account, at least in part, for the different effect on PMN recruitment. In addition, we show for the first time that VEGF + bFGF synergize to enhance monocyte and PMN recruitment during acute inflammation. Thus, these growth factors have important and, in part, distinct, proinflammatory properties, unique in that they can act, at least acutely, as positive regulators rather than as primary agonists of leukocyte recruitment. Their joint overexpression in many inflammatory conditions warrants that their role be investigated further in acute and chronic inflammatory diseases.

ACKNOWLEDGEMENTS

This work was supported by Grants MT-7684 and MGC-57081 from the Canadian Institutes of Health Research. S. I. Z. is the recipient of a fellowship from the IWK Health Centre. The authors are grateful for the excellent technical assistance of Ms. C. Jordan and Mr. D. Rowter. We also thank Drs. M. Miyasaka, L. Burkly, and T. B. Issekutz for gifts of mAb. We give our gratitude as well to Ms. Anne Woolaver for her excellent secretarial assistance.

Received December 6, 2005; revised March 2, 2006; accepted March 27, 2006.

REFERENCES

  1. Springer, T. A. (1995) Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration Annu. Rev. Physiol. 57,827-872[CrossRef][Medline]
  2. Pintavorn, P., Ballermann, B. J. (1997) TGF-ß and the endothelium during immune injury Kidney Int. 51,1401-1412[Medline]
  3. Lee, T. H., Avraham, H., Lee, S. H., Avraham, S. (2002) Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells J. Biol. Chem. 277,10445-10451[Abstract/Free Full Text]
  4. Tromp, S. C., oude Egbrink, M. G., Dings, R. P., van Velzen, S., Slaaf, D. W., Hillen, H. F., Tangelder, G. J., Reneman, R. S., Griffioen, A. W. (2000) Tumor angiogenesis factors reduce leukocyte adhesion in vivo Int. Immunol. 12,671-676[Abstract/Free Full Text]
  5. Ferrara, N., Davis-Smyth, T. (1997) The biology of vascular endothelial growth factor Endocr. Rev. 18,4-25[Abstract/Free Full Text]
  6. Griffioen, A. W., Molema, G. (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation Pharmacol. Rev. 52,237-268[Abstract/Free Full Text]
  7. Manabe, N., Oda, H., Nakamura, K., Kuga, Y., Uchida, S., Kawaguchi, H. (1999) Involvement of fibroblast growth factor-2 in joint destruction of rheumatoid arthritis patients Rheumatology (Oxford) 38,714-720[Medline]
  8. Kikuchi, K., Hoashi, T., Kanazawa, S., Tamaki, K. (2005) Angiogenic cytokines in serum and cutaneous lesions of patients with polyarteritis nodosa J. Am. Acad. Dermatol. 53,57-61[CrossRef][Medline]
  9. Redington, A. E., Roche, W. R., Madden, J., Frew, A. J., Djukanovic, R., Holgate, S. T., Howarth, P. H. (2001) Basic fibroblast growth factor in asthma: measurement in bronchoalveolar lavage fluid basally and following allergen challenge J. Allergy Clin. Immunol. 107,384-387[CrossRef][Medline]
  10. Weis, S. M., Cheresh, D. A. (2005) Pathophysiological consequences of VEGF-induced vascular permeability Nature 437,497-504[CrossRef][Medline]
  11. Detmar, M., Brown, L. F., Berse, B., Jackman, R. W., Elicker, B. M., Dvorak, H. F., Claffey, K. P. (1997) Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin J. Invest. Dermatol. 108,263-268[CrossRef][Medline]
  12. Sandner, P., Wolf, K., Bergmaier, U., Gess, B., Kurtz, A. (1997) Induction of VEGF and VEGF receptor gene expression by hypoxia: divergent regulation in vivo and in vitro Kidney Int. 51,448-453[Medline]
  13. Alagappan, V. K., McKay, S., Widyastuti, A., Garrelds, I. M., Bogers, A. J., Hoogsteden, H. C., Hirst, S. J., Sharma, H. S. (2005) Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells Cell Biochem. Biophys. 43,119-129[CrossRef][Medline]
  14. Pages, G., Pouyssegur, J. (2005) Transcriptional regulation of the vascular endothelial growth factor gene—a concert of activating factors Cardiovasc. Res. 65,564-573[CrossRef][Medline]
  15. Bikfalvi, A., Klein, S., Pintucci, G., Rifkin, D. B. (1997) Biological roles of fibroblast growth factor-2 Endocr. Rev. 18,26-45[Abstract/Free Full Text]
  16. Detmar, M., Brown, L. F., Schon, M. P., Elicker, B. M., Velasco, P., Richard, L., Fukumura, D., Monsky, W., Claffey, K. P., Jain, R. K. (1998) Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice J. Invest. Dermatol. 111,1-6[CrossRef][Medline]
  17. Dirkx, A. E., Oude Egbrink, M. G., Kuijpers, M. J., van der Niet, S. T., Heijnen, V. V., Bouma-ter Steege, J. C., Wagstaff, J., Griffioen, A. W. (2003) Tumor angiogenesis modulates leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion molecule expression Cancer Res. 63,2322-2329[Abstract/Free Full Text]
  18. Griffioen, A. W., Damen, C. A., Blijham, G. H., Groenewegen, G. (1996) Tumor angiogenesis is accompanied by a decreased inflammatory response of tumor-associated endothelium Blood 88,667-673[Abstract/Free Full Text]
  19. Zhang, H., Issekutz, A. C. (2002) Down-modulation of monocyte transendothelial migration and endothelial adhesion molecule expression by fibroblast growth factor: reversal by the anti-angiogenic agent SU6668 Am. J. Pathol. 160,2219-2230[Abstract/Free Full Text]
  20. Reinders, M. E., Sho, M., Izawa, A., Wang, P., Mukhopadhyay, D., Koss, K. E., Geehan, C. S., Luster, A. D., Sayegh, M. H., Briscoe, D. M. (2003) Proinflammatory functions of vascular endothelial growth factor in alloimmunity J. Clin. Invest. 112,1655-1665[CrossRef][Medline]
  21. Kanazawa, S., Tsunoda, T., Onuma, E., Majima, T., Kagiyama, M., Kikuchi, K. (2001) VEGF, basic-FGF, and TGF-ß in Crohn’s disease and ulcerative colitis: a novel mechanism of chronic intestinal inflammation Am. J. Gastroenterol. 96,822-828[Medline]
  22. Zittermann, S. I., Issekutz, A. C. (2006) Basic fibroblast growth factor (bFGF; FGF-2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression Am. J. Pathol. 168,835-846[Abstract/Free Full Text]
  23. Belgore, F., Lip, G. Y., Blann, A. D. (2003) Basic fibrobrast growth factor induces the secretion of vascular endothelial growth factor by human aortic smooth muscle cells but not by endothelial cells Eur. J. Clin. Invest. 33,833-839[CrossRef][Medline]
  24. Issekutz, A. C., Issekutz, T. B. (1993) Quantitation and kinetics of blood monocyte migration to acute inflammatory reactions, and IL-1 {alpha}, tumor necrosis factor-{alpha}, and IFN-{gamma} J. Immunol. 151,2105-2115[Abstract]
  25. Walter, U. M., Ayer, L. M., Wolitzky, B. A., Wagner, D. D., Hynes, R. O., Manning, A. M., Issekutz, A. C. (1997) Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse Hybridoma 16,249-257[Medline]
  26. Walter, U. M., Ayer, L. M., Manning, A. M., Frenette, P. S., Wagner, D. D., Hynes, R. O., Wolitzky, B. A., Issekutz, A. C. (1997) Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin Hybridoma 16,355-361[Medline]
  27. May, M. J., Entwistle, G., Humphries, M. J., Ager, A. (1993) VCAM-1 is a CS1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium J. Cell Sci. 106,109-119[Abstract]
  28. Tamatani, T., Miyasaka, M. (1990) Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells Int. Immunol. 2,165-171[Abstract/Free Full Text]
  29. Tamatani, T., Kotani, M., Miyasaka, M. (1991) Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies Eur. J. Immunol. 21,627-633[Medline]
  30. Issekutz, T. B., Wykretowicz, A. (1991) Effect of a new monoclonal antibody, TA-2, that inhibits lymphocyte adherence to cytokine-stimulated endothelium in the rat J. Immunol. 147,109-116[Abstract]
  31. Udaka, K., Takeuchi, Y., Movat, H. Z. (1970) Simple method for quantitation of enhanced vascular permeability Proc. Soc. Exp. Biol. Med. 133,1384-1387[Medline]
  32. Issekutz, A. C. (1981) Vascular responses during acute neutrophilic inflammation. Their relationship to in vivo neutrophil emigration Lab. Invest. 45,435-441[Medline]
  33. Issekutz, T. B. (1992) Inhibition of lymphocyte endothelial adhesion and in vivo lymphocyte migration to cutaneous inflammation by TA-3, a new monoclonal antibody to rat LFA-1 J. Immunol. 149,3394-3402[Abstract]
  34. Issekutz, T. B. (1995) In vivo blood monocyte migration to acute inflammatory reactions, IL-1 {alpha}, TNF-{alpha}, IFN-{gamma}, and C5a utilizes LFA-1, Mac-1, and VLA-4. The relative importance of each integrin J. Immunol. 154,6533-6540[Abstract]
  35. Issekutz, T. B., Miyasaka, M., Issekutz, A. C. (1996) Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation J. Exp. Med. 183,2175-2184[Abstract/Free Full Text]
  36. Marshall, D., Haskard, D. O. (2003) Quantifying inflammation in vivo using radiolabeled antibodies and leukocytes Methods Mol. Biol. 225,273-282[Medline]
  37. Issekutz, A. C., Issekutz, T. B. (2002) The role of E-selectin, P-selectin, and very late activation antigen-4 in T lymphocyte migration to dermal inflammation J. Immunol. 168,1934-1939[Abstract/Free Full Text]
  38. Barleon, B., Sozzani, S., Zhou, D., Weich, H. A., Mantovani, A., Marme, D. (1996) Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1 Blood 87,3336-3343[Abstract/Free Full Text]
  39. Watanabe, H., Mamelak, A. J., Wang, B., Howell, B. G., Freed, I., Esche, C., Nakayama, M., Nagasaki, G., Hicklin, D. J., Kerbel, R. S., Sauder, D. N. (2004) Anti-vascular endothelial growth factor receptor-2 (Flk-1/KDR) antibody suppresses contact hypersensitivity Exp. Dermatol. 13,671-681[CrossRef][Medline]
  40. Yamashita, A., Yonemitsu, Y., Okano, S., Nakagawa, K., Nakashima, Y., Irisa, T., Iwamoto, Y., Nagai, Y., Hasegawa, M., Sueishi, K. (2002) Fibroblast growth factor-2 determines severity of joint disease in adjuvant-induced arthritis in rats J. Immunol. 168,450-457[Abstract/Free Full Text]
  41. Issekutz, A. C. (1981) Effect of vasoactive agents on polymorphonuclear leukocyte emigration in vivo Lab. Invest. 45,234-240[Medline]
  42. Pasieka, Z., Stepien, H., Komorowski, J., Kolomecki, K., Kuzdak, K. (2003) Evaluation of the levels of bFGF, VEGF, sICAM-1, and sVCAM-1 in serum of patients with thyroid cancer Recent Results Cancer Res. 162,189-194[Medline]
  43. Guo, Z. H., Mei, H., Huang, J., Li, S. Y. (2003) Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression in superficial transitional cell bladder carcinoma Ai Zheng 22,307-309[Medline]
  44. Usui, T., Ishida, S., Yamashiro, K., Kaji, Y., Poulaki, V., Moore, J., Moore, T., Amano, S., Horikawa, Y., Dartt, D., Golding, M., Shima, D. T., Adamis, A. P. (2004) VEGF164(165) as the pathological isoform: differential leukocyte and endothelial responses through VEGFR1 and VEGFR2 Invest. Ophthalmol. Vis. Sci. 45,368-374[Abstract/Free Full Text]
  45. Croll, S. D., Ransohoff, R. M., Cai, N., Zhang, Q., Martin, F. J., Wei, T., Kasselman, L. J., Kintner, J., Murphy, A. J., Yancopoulos, G. D., Wiegand, S. J. (2004) VEGF-mediated inflammation precedes angiogenesis in adult brain Exp. Neurol. 187,388-402[CrossRef][Medline]
  46. Kim, I., Moon, S. O., Kim, S. H., Kim, H. J., Koh, Y. S., Koh, G. Y. (2001) Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-{kappa} B activation in endothelial cells J. Biol. Chem. 276,7614-7620[Abstract/Free Full Text]
  47. Radisavljevic, Z., Avraham, H., Avraham, S. (2000) Vascular endothelial growth factor up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/nitric oxide pathway and modulates migration of brain microvascular endothelial cells J. Biol. Chem. 275,20770-20774[Abstract/Free Full Text]
  48. Chuluyan, H. E., Issekutz, A. C. (1993) The VLA-4 integrin can mediate CD11/CD18 independent transendothelial migration of human monocytes J. Clin. Invest. 92,2768-2777[Medline]
  49. Chuluyan, H. E., Osborn, L., Lobb, R., Issekutz, A. C. (1995) Domains 1 and 4 of vascular cell adhesion molecule-1 (CD106) both support very late activation antigen-4 (CD49d/CD29)-dependent monocyte transendothelial migration J. Immunol. 155,3135-3144[Abstract]
  50. Issekutz, A. C., Issekutz, T. B. (1995) Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms J. Exp. Med. 181,1197-1203[Abstract/Free Full Text]
  51. Meerschaert, J., Furie, M. B. (1994) Monocytes use either CD11/CD18 or VLA-4 to migrate across human endothelium in vitro J. Immunol. 152,1915-1926[Abstract]
  52. Zhang, H., Issekutz, A. C. (2001) Growth factor regulation of neutrophil-endothelial cell interactions J. Leukoc. Biol. 70,225-232[Abstract/Free Full Text]
  53. Marumo, T., Schini-Kerth, V. B., Busse, R. (1999) Vascular endothelial growth factor activates nuclear factor-{kappa}B and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells Diabetes 48,1131-1137[Abstract]
  54. Paludan, S. R. (2000) Synergistic action of pro-inflammatory agents: cellular and molecular aspects J. Leukoc. Biol. 67,18-25[Abstract]

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