Monocyte-Fibronectin Interactions, Via ␣51 Integrin, Induce Expression of CXC Chemokine-Dependent Angiogenic Activity1 Eric S. White,* Donna L. Livant,† Sonja Markwart,† and Douglas A. Arenberg2* Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serumdifferentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene ␣, and growth-related oncogene ␥) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MGFNⴚ) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MGFNⴚ revealed a significant decrease in GRO-␣ and GRO-␥ compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via ␣51 integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via ␣51 integrin, promotes CXC chemokinedependent angiogenic activity from monocytes. The Journal of Immunology, 2001, 167: 5362–5366.
A
ngiogenesis is a central process in numerous pathologic and normal conditions, such as malignancy (1, 2), rheumatoid arthritis (3, 4) and wound repair (5, 6). Many investigators have drawn parallels between the processes of wound repair and solid tumor growth (7, 8). Each process is characterized, to varying extents, by fibrin clot formation, deposition of primordial extracellular matrix, proliferation of epithelium, and neovascularization. Although monocyte-derived macrophages are known to be critically important cells in wound repair (9, 10), their role in tumor growth remains controversial. However, it is reasonable to hypothesize a similar role for macrophages in promoting tumor growth (11, 12). We and others have demonstrated that tumors are infiltrated with monocyte-derived macrophages, and that this infiltration is mediated by members of the CC chemokine family (13, 14). Moreover, we have recently demonstrated that soluble factors derived from non-small cell lung cancer (NSCLC)3 cell lines induce angiogenic activity from monocytes (15).
*Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and †Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109 Received for publication June 6, 2001. Accepted for publication August 23, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported, in part, by National Institutes of Health Grants CA72543 (to D.A.A.) and T32HL07749-07 (to E.S.W.) and by American Lung Association Research Grant RG-065-N (to D.A.A.). D.A.A. is a Sidney Kimmel Foundation Scholar. 2 Address correspondence and reprint requests to Dr. Douglas A. Arenberg, Division of Pulmonary and Critical Care, University of Michigan Medical Center, 6301 MSRB III, Box 0642, 1150 West Medical Center Drive, Ann Arbor, MI 48109. E-mail address:
[email protected] 3 Abbreviations used in this paper: NSCLC, non-small cell lung cancer; CM, conditioned medium; ECC, endothelial cell chemotaxis; ENA, epithelial-neutrophil-activating peptide; GRO, growth-related oncogene; VEGF, vascular endothelial growth
Copyright © 2001 by The American Association of Immunologists
In this study, we hypothesize a role for cell-matrix interactions in inducing angiogenic activity from monocytes. We speculate that when monocytes are recruited to a tumor, they encounter an extracellular matrix environment that promotes the development of an angiogenic phenotype and neovascularization. To test this hypothesis, we cultured peripheral blood monocytes on varying extracellular matrix proteins including collagen I, fibronectin, laminin, and Matrigel (basement membrane extract). In addition, monocytes were cultured in the presence of autologous serum on tissue culture plastic. Conditioned medium (CM) was generated from monocytes cultured in the various conditions, and endothelial cell chemotaxis (ECC) assays were performed to assess angiogenic potential. We found that monocytes cultured on Matrigel or fibronectin displayed the greatest degree of angiogenic activity. When monocytes are cultured on fibronectin-depleted Matrigel in comparison to monocytes cultured on Matrigel, there is a significantly decreased angiogenic activity of the CM, suggesting that fibronectin is responsible in part for the induction of angiogenic activity seen from Matrigel-cultured monocytes. Subsequent ELISA of supernatants from monocytes cultured on Matrigel showed a marked up-regulation of the angiogenic CXC chemokines IL-8 (IL-8 or CXCL8), epithelial-neutrophil-activating peptide 78 (ENA-78 or CXCL5), and growth-related oncogenes ␣ and ␥ (GRO-␣ or CXCL1 and GRO-␥ or CXCL3, respectively), but not of vascular endothelial growth factor (VEGF). Expression of these chemokines was decreased when monocytes were cultured on fibronectin-depleted Matrigel. These data support the hypothesis that monocytes, upon extravasation into the tumor extracellular matrix, develop a phenotype characterized by increased expression of angiogenic activity. factor; MGFN⫺, fibronectin-depleted Matrigel; hpf, high-power field; TCP, tissue culture plastic. 0022-1767/01/$02.00
The Journal of Immunology
Materials and Methods Cell lines Human peripheral blood monocyte medium was RPMI 1640 supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 1% L-glutamine, and 25 M HEPES buffer. Human dermal microvascular endothelial cells (HMEC-1) were a generous gift from Dr. E. Ades and F. J. Candal (Centers for Disease Control, National Center for Infectious Diseases, Atlanta, GA) and Dr. T. Lawley (Emory University, Atlanta, GA). Human microvascular endothelial cell lines HMVEC (Cell Systems, Kirkland, WA) and HMEC-1 were maintained per the supplier’s recommendations. Media were changed in all cell lines every 48–72 h, and cells were maintained at 37°C with 5% CO2.
Antibodies Polyclonal rabbit anti-human IL-8 was prepared as previously described. Polyclonal goat anti-human ENA-78, goat anti-human GRO-␣, and goat anti-human VEGF were purchased from R&D Systems (Minneapolis, MN). Polyclonal rabbit anti-human GRO-␥ was purchased from PeproTech (Rocky Hill, NJ). The polyclonal anti-human VEGF Ab is specific for VEGF121 and VEGF165, the predominant isoforms produced by macrophages (17). Some Abs were biotinylated for use in ELISA detection steps as described (18). Goat anti-monokine induced by IFN-␥, and goat antiIFN-␥-inducible protein 10 were obtained from R&D Systems. Rabbit antihuman CXCR2 was a kind gift from Dr. R. Strieter (University of California, Los Angeles, School of Medicine, Los Angeles, CA). Preimmune rabbit and goat IgG was purchased from R&D Systems.
Tissue culture coating We coated 24-well plates (Nalge Nunc, Naperville, IL) with fibronectin (BD PharMingen, San Diego, CA) at 100 g/ml, Matrigel or fibronectindepleted Matrigel (MGFN⫺; Collaborative Biomedical Products, Bedford, MA) at 1 mg/ml, collagen I at 50 g/ml in 0.02 N acetic acid, or laminin (BD PharMingen) at 100 g/ml. Supplier’s recommendations were used to determine working concentrations of each matrix protein. Matrigel was depleted of fibronectin by immunoprecipitation with protein G-Sepharose (Sigma-Aldrich, St. Louis, MO). Mouse anti-human fibronectin Ab was bound to protein G-Sepharose and the protein G-Ab mixture was incubated with Matrigel on a rocker plate for 3 h at 4°C. Following incubation, the Sepharose was removed by centrifugation. Fibronectin depletion was confirmed by immunoblot analysis (data not shown). Tissue-culture treated 24-well plates were coated with 100 l of the appropriate matrix protein to each well, incubated for 1 h at room temperature, rinsed with sterile medium, and dried in a laminar flow hood. Plates could be kept at 4°C for up to 1 wk after coating.
Monocyte isolation After informed consent was obtained, whole blood (anticoagulated with 1000 U of heparin per 60 ml) was obtained by venipuncture from healthy, nonsmoking donors, and monocytes were obtained by Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density centrifugation and adherence purification as previously described (15). For some conditions, monocytes were cultured in 2% autologous serum (saved at the time of initial phlebotomy). After 48 h, cells were washed twice with PBS and serum-free medium was replaced. This medium was then harvested after an additional 24 h to generate CM from monocytes differentiated for 2 days in each of the conditions above. All samples were stored at ⫺20°C until used in assays. For some experiments, freshly isolated monocytes were incubated with the peptides PHSCN or HSPNC (14 M) for 1 h at 4°C before adherence purification on fibronectin-coated plates.
5363 Membranes were fixed in methanol, stained with a modified Wright-Giemsa stain, and cells that had migrated through the membrane were counted in five high-power fields (hpf; ⫻200). Results were expressed as the mean number of endothelial cells that had migrated per hpf ⫾ SEM. Each sample was assessed in triplicate. Each experiment was performed a minimum of three times.
Chemokine ELISA Antigenic CXC chemokines (IL-8, ENA-78, GRO-␣, GRO-␥) and VEGF were quantitated using a double ligand method previously described (20). Standards were prepared as half-log dilutions of purified recombinant protein, from 100 to 0.001 ng/ml per well.
Statistical analyses All generated data were compared by Student’s t test for unpaired observations or one-way ANOVA with Bonferroni posttest analysis where indicated, and were considered significant if p ⬍ 0.05. Data were analyzed on a Dell computer using the StatView 5.0.1 statistical software package (SAS Institute, Cary, NC) or GraphPad Prism 3.0 (GraphPad, San Diego, CA).
Results Culture of monocytes on matrix proteins induces increased endothelial cell chemotactic activity We hypothesized that monocytes encountering the tumor microenvironment would be exposed to conditions that led to increased angiogenic activity. To test this, we used 24-h CM for ECC from monocytes that had been cultured on different extracellular matrix proteins. Matrigel, a mixture of basement membrane proteins, was chosen as a simulation for the type of primordial matrix likely to be encountered in a tumor. CM from monocytes cultured on Matrigel exhibited increased endothelial cell chemotactic activity (87 ⫾ 6 cells/hpf) as compared with cells cultured in 2% autologous serum (40 ⫾ 3 cells/hpf), collagen I (39 ⫾ 2 cells/hpf), or laminin (38 ⫾ 5 cells/hpf; p ⬍ 0.0001 for all comparisons; Fig. 1). All conditions demonstrated increased chemotactic activity compared with negative medium control (14 ⫾ 4 cells/hpf; p ⬍ 0.0001 for all conditions). Interestingly, CM from monocytes cultured on fibronectin displayed chemotactic activity similar to monocytes cultured on Matrigel (69 ⫾ 7 cells/hpf; p ⫽ 0.3; Fig. 1). This angiogenic activity was similarly greater than that seen from CM from cells cultured on collagen, laminin, or with autologous serum ( p ⬍ 0.01 for each condition). To exclude the possibility that the ECC seen from monocytes cultured on Matrigel was due to angiogenic factors leached from the Matrigel, we performed ECC assays on media taken from cell-free wells that were coated with
Peptide synthesis The peptides PHSCN (acetyl-Pro-His-Ser-Cys-Asn-NH2) and HSPNC (acetyl-His-Ser-Pro-Asn-Cys-NH2) were synthesized using standard F-moc/t-butyl protection strategies (19). Both peptides were synthesized at 100-mol scales using a Rainin Symphony multiple peptide synthesizer by the University of Michigan Protein and Carbohydrate Structure Facility. Peptides were then purified using HPLC. Peptide structures were confirmed by mass spectrometry and amino acid analysis (data not shown).
ECC assay ECC assays were performed in 12-well, blind-well chemotaxis chambers (NeuroProbe, Gaithersburg, MD) as previously described (15). After the appropriate time for chemotaxis had elapsed, membranes were removed and nonadherent cells were removed by scraping with a rubber policeman.
FIGURE 1. ECC to CM from monocytes cultured on various extracellular matrix proteins, Matrigel, or in the presence of 2% autologous serum. Control refers to protein-free culture media alone. ⴱ, Comparison between Matrigel-cultured monocyte CM and control, or serum-, laminin-, or collagen-cultured monocyte CM. ⴱⴱ, Comparison between fibronectin-cultured monocyte CM and control, or serum-, laminin-, or collagen-cultured monocytes. The difference between Matrigel-cultured monocyte CM and fibronectin-cultured monocyte CM was not significant (p ⫽ 0.3). All experiments were performed three times.
5364
MONOCYTE-DERIVED CXC CHEMOKINES INDUCED BY FIBRONECTIN demonstrated a trend toward decreased expression in CM from monocytes cultured on MGFN⫺ compared with CM from monocytes cultured on Matrigel ( p ⫽ 0.06 for each chemokine). Furthermore, angiogenic CXC chemokine levels were significantly increased in both conditions compared with CM from monocytes cultured on TCP ( p ⬍ 0.05 for all conditions; Table I). VEGF levels were below the lower limit of detection of the ELISA (⬍0.01 ng/ml) for all samples (Table I).
FIGURE 2. ECC to CM from monocytes cultured on Matrigel (MG) or MGFN⫺. Control refers to protein-free culture media alone. ⴱ, Comparison between Matrigel and MGFN⫺. Both Matrigel and MGFN⫺ were significantly greater than media control (p ⬍ 0.001). Data shown are representative of three repeated experiments.
Matrigel. This media did not induce any endothelial cell chemotactic activity compared with negative control (data not shown). Depletion of fibronectin from Matrigel reduces the induction of monocyte-derived angiogenic activity To determine whether the increased endothelial cell chemotactic activity seen from monocytes cultured on Matrigel was due to fibronectin within Matrigel, we first depleted Matrigel of fibronectin by immunoprecipitation. We then collected 24-h CM from monocytes cultured on either Matrigel or MGFN⫺ and performed ECC assays. We found that ECC to CM from monocytes cultured on MGFN⫺ demonstrated significantly reduced angiogenic activity when compared with CM from monocytes cultured on Matrigel (74 ⫾ 3 cells/hpf vs 92 ⫾ 3 cells/hpf; p ⫽ 0.0001; Fig. 2). This suggested that at least part of the ability of Matrigel to induce angiogenic activity from monocytes was due to the presence of fibronectin within Matrigel. Angiogenic CXC chemokines, but not VEGF, are increased in CM from monocytes cultured on Matrigel We have previously shown that CXC chemokines are important mediators of angiogenesis in NSCLC (21, 22). Furthermore, monocyte-derived macrophages are a potent source of these chemokines (15). Therefore, we hypothesized that the increased angiogenic activity seen in CM from monocytes cultured on Matrigel was due to increased expression of angiogenic CXC chemokines. To test this hypothesis, we performed ELISA for the angiogenic CXC chemokines as well as VEGF on CM from monocytes cultured on tissue culture plastic (TCP), Matrigel, or MGFN⫺. We found that CXC chemokine expression from monocytes cultured on Matrigel was markedly increased compared with monocytes cultured on TCP. Subsequently, we observed that monocytes cultured on MGFN⫺ demonstrated a decreased expression of CXC chemokines compared with monocytes cultured on Matrigel. GRO-␣ and GRO-␥ levels in CM from monocytes cultured on MGFN⫺ were significantly decreased compared with CM from monocytes cultured on Matrigel (Table I). IL-8 and ENA-78 levels
ECC to CM from monocytes cultured on fibronectin is diminished in the presence of neutralizing Abs to angiogenic CXC chemokines, but not to neutralizing anti-VEGF Abs The significance of decreased endothelial cell chemotactic activity in the CM of monocytes cultured on MGFN⫺ is unclear. Although statistically significant, the magnitude of reduction may not be biologically significant. This may be related to the myriad growth factors and other proteins within Matrigel that may influence monocytes. To better define the role of fibronectin in mediating the increase in monocyte-derived angiogenic activity, we focused on monocyte-fibronectin interactions. The preceding data demonstrated increased CXC chemokine expression from monocytes cultured on Matrigel, which was partly dependent on the presence of fibronectin within Matrigel. To determine whether CXC chemokines are responsible for the increased angiogenic activity seen in CM from monocytes cultured on fibronectin, we performed ECC in the presence of neutralizing Abs to IL-8, ENA-78, GRO-␣, GRO-␥, or control Abs using CM generated from monocytes cultured on fibronectin as the stimulus. We found that ECC was significantly reduced in the presence of neutralizing Abs to CXC chemokines compared with control ( p ⬍ 0.001 for all conditions; Fig. 3A). In contrast, neutralizing Ab to VEGF did not decrease ECC compared with control IgG (data not shown). To further demonstrate the role of angiogenic CXC chemokines in this system, we pretreated endothelial cells with neutralizing Ab to CXCR2, the putative receptor for the angiogenic CXC chemokines, or control IgG and performed ECC to CM generated by monocytes cultured on fibronectin. Blockade of CXCR2 resulted in a significant reduction in ECC compared with IgG control (46 ⫾ 3 cells/hpf vs 86 ⫾ 3 cells/hpf; p ⬍ 0.001; Fig. 3B), consistent with the above findings for neutralization of the ligands. This suggested that the observed ECC was mediated in part through this receptor. Monocyte-derived CXC chemokine-dependent angiogenic activity induced by fibronectin is mediated through ␣51 integrin Monocyte binding to fibronectin is mediated primarily by ligation of the ␣51 integrin (24) through both the PHSRN (Pro-His-SerArg-Asn) and RGD (Arg-Gly-Asp) sequences located in the 9th and 10th type III repeat of fibronectin, respectively (25). These two sequences act in a synergistic fashion to increase the affinity of ␣51 integrin for fibronectin (25). Interestingly, the PHSRN pentapeptide alone is sufficient to stimulate cellular invasion into basement membranes. Furthermore, PHSRN has recently been shown
Table I. Effect of culture substrate on chemokine expression from peripheral blood monocytesa Condition
TCP FN MG MGFN⫺
IL-8 (ng/ml)
ENA-78 (ng/ml)
GRO-␣ (ng/ml)
GRO-␥ (ng/ml)
VEGF (ng/ml)
20.7 ⫾ 0.9 142.4 ⫾ 11.7 148.8 ⫾ 37.5 50.9 ⫾ 10.2
1.1 ⫾ 0.2 18.8 ⫾ 9.5 119.4 ⫾ 25.5 48.5 ⫾ 10.9
0.4 ⫾ 0.1 3.2 ⫾ 0.2 12.1 ⫾ 2.4 4.6 ⫾ 1.4*
4.2 ⫾ 1.0 39.6 ⫾ 2.6 115.5 ⫾ 6.7 8.8 ⫾ 1.4**
⬍0.01b ⬍0.01b ⬍0.01b ⬍0.01b
a ELISA analysis of CM from monocytes cultured on TCP, fibronectin (FN), Matrigel (MG), or MGFN⫺ for angiogenic CXC chemokines and VEGF. Results are expressed as nanograms per milliliter ⫾ SEM. ⴱ, p ⬍ 0.05 for the comparison of GRO-␣ levels in CM from monocytes cultured on Matrigel compared to CM from monocytes cultured on MGFN⫺. ⴱⴱ, p ⬍ 0.001 for the comparison of GRO-␥ levels in CM from monocytes cultured on Matrigel compared to CM from monocytes cultured on MGFN⫺. b Detection limit of the assay. Data are representative of three separate experiments performed in triplicate.
The Journal of Immunology
5365 these CM for angiogenic CXC chemokines revealed a significant decrease in CXC chemokine expression from monocytes cultured on fibronectin in the presence of PHSCN compared with monocytes cultured on fibronectin in the presence of HSPNC (Fig. 5). To ensure that the difference in angiogenic activity and chemokine level was not due to decreased monocyte adherence or cell death, all wells were evaluated for viability via trypan blue exclusion as well as for total cell count per well. There was no significant difference between the HSPNC-treated group and the PHSCN-treated group (data not shown).
Discussion
FIGURE 3. A, ECC to CM from monocytes cultured on fibronectin. Control refers to protein-free culture media alone. Each condition on the abscissa refers to the addition to CM of control Ab (IgG) or chemokinespecific neutralizing Ab. In each case, the addition of neutralizing Ab significantly decreased ECC compared with control Ab (p ⬍ 0.001 for each condition). Data shown are representative of three separate experiments. B, ECC to CM from monocytes cultured on fibronectin. Control refers to protein-free culture media alone. Before performing chemotaxis assays, endothelial cells were incubated with neutralizing Ab to CXCR2, the putative receptor for angiogenic CXC chemokines, or control IgG. Endothelial cells pretreated with ␣CXCR2 Ab demonstrated a 46% decrease in chemotaxis compared with control IgG-treated endothelial cells. Data are representative of three repeated experiments.
to significantly promote wound healing in diabetic mice (26). The peptide sequence PHSCN (Pro-His-Ser-Cys-Asn) has been shown to be a competitive inhibitor of ␣51-PHSRN binding (27). To determine whether fibronectin-␣51 interactions were responsible for the increased monocyte-derived angiogenic activity observed, we preincubated monocytes with PHSCN or HSPNC (scrambled control peptide) before culturing monocytes on fibronectin. CM was harvested and used in ECC and ELISA. We found that compared with the control peptide, monocytes preincubated with PHSCN then cultured on fibronectin induced significantly less endothelial cell chemotactic activity (58 ⫾ 4 cells/hpf vs 84 ⫾ 6 cells/hpf; p ⬍ 0.001; Fig. 4). Additionally, ELISA analysis of
FIGURE 4. ECC to CM from monocytes cultured on fibronectin. Before plating on fibronectin, monocytes were preincubated with a competitive peptide inhibitor of ␣51 binding (PHSCN) or control peptide (HSPNC). Compared with control peptide-treated monocytes, PHSCNtreated monocytes induced 40% less ECC. Data shown are representative of two repeated experiments.
To determine how monocytes might affect angiogenesis in a tumor environment, we studied the interaction between peripheral blood monocytes and extracellular matrix proteins. Although we were primarily interested in modeling the tumor environment, similar monocyte-matrix interactions might be assumed to occur in wounds (28) or in other conditions of tissue inflammation (11). Upon extravasation from blood vessels, monocytes initially encounter extracellular matrix proteins common in the tumor environment (11, 29) such as collagen, fibronectin, and laminin. We postulated that this extracellular matrix would induce a change in monocyte phenotype to increase expression of angiogenic factors. Although we did observe increased expression of angiogenic activity in CM from monocytes cultured on collagen, laminin, and fibronectin compared with negative control, the greatest increase in angiogenic activity was seen in CM from monocytes cultured on fibronectin. Therefore, we chose to focus on fibronectin-monocyte interactions. We found that the angiogenic phenotype of monocytes cultured on fibronectin was characterized by CXC chemokine-dependent endothelial cell chemotactic activity. We have previously shown that angiogenic CXC chemokines are important regulators of angiogenic activity in NSCLC (21, 30). Although we have found that many NSCLC cell lines spontaneously produce CXC chemokines, our previous data suggested that other cells within the tumor might also be important sources of these molecules (21). In this study, our in vitro findings support the notion that a stromal environment analogous to what might be found in a tumor may influence infiltrating cell types to create an angiogenic milieu by increased elaboration of the angiogenic CXC chemokines. Interestingly, in addition to cell-matrix interactions that induce monocyte-derived angiogenic activity, we have found that malignant cells produce soluble factors that induce expression of angiogenic factors from monocytes as well (15). We found that increased angiogenic activity of monocytes cultured on fibronectin could be inhibited by blocking ␣51-PHSRN
FIGURE 5. ELISA analysis of CM from monocytes cultured on fibronectin in the presence of a competitive peptide inhibitor of ␣51 binding (PHSCN) or control peptide (HSPNC). For each chemokine, comparison is made between PHSCN- and HSPNC-treated monocytes. Results are expressed as nanograms per milliliter chemokine ⫾ SEM. In each case, PHSCN-treated monocytes elaborated significantly less chemokine than HSPNC-treated monocytes. Data are representative of three separate experiments.
5366
MONOCYTE-DERIVED CXC CHEMOKINES INDUCED BY FIBRONECTIN
ligation using the PHSCN pentapeptide. It has been suggested that vascular endothelial cells participate in neovascularization using a number of integrins, including ␣51, as well as ␣41 and ␣v3 (31, 32). In vivo experiments have demonstrated that fibronectin binds to the ␣51 integrin on endothelial cells to modulate tumor angiogenesis and that blocking this interaction results in decreased tumor growth and vascularity (33). Results of the present study demonstrate that monocytes also interact with fibronectin through the ␣51 integrin leading to an increased expression of angiogenic factors. The potentially dual role for ␣51 integrin in promoting angiogenesis makes it an attractive target for antitumor therapy. Indeed, we have shown that blocking ␣51-PHSRN interactions with the peptide antagonist PHSCN leads to reduced tumor growth and metastasis of the MATLyLu prostate cancer cell line (27). Previous studies have demonstrated that fibronectin also initiates cell signaling events through ligation of the ␣41 integrin (34). This may be of relevance because blocking fibronectin binding to ␣51 integrin on monocytes using the inhibitory peptide PHSCN did not completely block elaboration of angiogenic activity in CM, suggesting an alternate pathway by which fibronectin induces the production of monocyte-derived angiogenic activity. Previous authors have suggested that solid tumors are composed of two separate compartments: the malignant cells and the tumor stroma consisting of extracellular matrix proteins and host cells (7). Recently, attention has been focused on the deposited extracellular matrix as a mediator of tumor growth and metastases (35). Additionally, evidence suggests that the extracellular matrix may play an active role in promoting resistance to chemotherapy and preventing tumor apoptosis (36). The role of extracellular matrix in tumor growth and metastasis has not yet been fully elucidated, but studies suggest that it provides a framework for malignant cells and host cells to interact (36). In wounds, inflammatory cells such as macrophages and platelets are known to interact with the extravasated plasma proteins and tissue matrix proteins to promote wound healing. In a similar fashion, monocyte-derived macrophages may interact with tumor extracellular matrix proteins to promote inflammatory cell recruitment, angiogenesis, and growth factor expression. This response may serve to promote tumor growth and metastasis. In summary, we have found that monocytes, upon adherence to fibronectin, develop a proangiogenic phenotype that depends upon angiogenic CXC chemokines. This increase is mediated by recognition of the PHSRN sequence of fibronectin via ␣51 integrin. The finding that integrin binding can lead to chemokine expression and angiogenic phenotype is a novel aspect of our current study.
References 1. Folkman, J., and R. Cotran. 1976. Relation of vascular proliferation to tumor growth. Int. Rev. Exp. Pathol. 16:207. 2. Hanahan, D., and J. Folkman. 1996. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353. 3. Koch, A. E., L. A. Harlow, G. K. Haines, E. P. Amento, E. N. Unemori, W. L. Wong, R. M. Pope, and N. Ferrara. 1994. Vascular endothelial growth factor: a cytokine modulating endothelial function in rheumatoid arthritis. J. Immunol. 152:4149. 4. Jackson, J. R., M. P. Seed, C. H. Kircger, D. A. Willoughby, and J. D. Winkler. 1997. The codependence of angiogenesis and chronic inflammation. FASEB J. 11:457. 5. Koch, A. E., M. A. Litvak, J. C. Burrows, and P. J. Polverini. 1992. Decreased monocyte-mediated angiogenesis in scleroderma. Clin. Immunol. Immunopathol. 64:153. 6. Leibovich, S. J., and D. M. Weisman. 1988. Macrophages, wound repair and angiogenesis. Prog. Clin. Biol. Res. 266:1131. 7. Dvorak, H. F. 1986. Wounds that do not heal. N Engl. J. Med. 315:1650. 8. Whalen, G. F. 1990. Solid tumors and wounds: transformed cells misunderstood as injured tissue? Lancet 336:1489.
9. DiPietro, L. A. 1995. Wound healing: the role of the macrophage and other immune cells. Shock 4:233. 10. Arnold, F., and D. C. West. 1991. Angiogenesis in wound healing. Pharmacol. Ther. 52:407. 11. Polverini. P. J. 1996. How the extracellular matrix and macrophages contribute to angiogenesis-dependent diseases. Eur. J. Cancer 32A:2430. 12. Polverini, P, J., and S. J. Leibovich. 1984. Induction of neovasularization in vivo and endothelial cell proliferation in vitro by tumor-associated macrophages. Lab. Invest. 51:635. 13. Orre, M., and P. A. Rogers. 1999. Macrophages and microvessel density in tumors of the ovary. Gynecol. Oncol. 73:47. 14. Arenberg, D. A., M. P. Keane, B. DiGiovine, S. L. Kunkel, S. R. B. Strom, M. D. Burdick, M. D. Iannettoni, and R. M. Strieter. 2000. Macrophage infiltration in human non-small cell lung cancer: the role of CC chemokines. Cancer Immunol. Immunother. 49:63. 15. White, E. S., S. R. Strom, N. L. Wys, and D. A. Arenberg. 2001. Non-small cell lung cancer cells induce monocytes to increase expression of angiogenic activity. J. Immunol. 166:7549. 16. Strieter, R. M., K. Kasahara, R. M. Allen, T. J. Standiford, M. W. Rolfe, F. S. Becker, S. W. Chensue, and S. L. Kunkel. 1992. Cytokine-induced neutrophil-derived interleukin-8. Am. J. Pathol. 141:397. 17. Kovacs, Z., K. Ikezaki, K. Samoto, T. Inamura, and M. Fukui. 1996. VEGF and flt: expression time kinetics in rat brain infarct. Stroke 27:1865. 18. Burdick, M. D., S. L. Kunkel, P. M. Lincoln, C. A. Wilke, and R. M. Strieter. 1993. Specific ELISAs for the detection of human macrophage inflammatory protein-1␣ and . Immunol. Invest. 22:441. 19. Atherton, E., and R. C. Sheppard. 1989. Solid Phase Peptide Synthesis: A Practical Approach. IRL, Oxford. 20. Arenberg, D. A., S. L. Kunkel, P, J, Polverini, S. B. Morris, M. D. Burdick, M. C. Glass, D. T. Taub, M. D. Iannettoni, R. I. Whyte, and R. M. Strieter. 1996. Interferon-␥-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184:981. 21. Arenberg, D. A., M. P. Keane, B. DiGiovine, S. L. Kunkel, S. B. Morris, Y. Y. Xue, M. D. Burdick, M. C. Glass, M. D. Iannettoni, and R. M. Strieter. 1998. Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer. J. Clin Invest. 102:465. 22. Arenberg, D. A., S. L. Kunkel, P. J. Polverini. M. Glass, M. D. Burdick, and R. M. Strieter. 1996. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J. Clin. Invest. 97:2792. 23. Addison, C., D. Arenberg, S. Morris, Y. Xue, M. Burdick, M. Mulligan, M. Iannettoni, and R. Strieter. 2000. The CXC chemokine, monokine induced by interferon ␥ (MIG), inhibits non-small cell lung cancer (NSCLC) tumor growth and metastasis. Hum. Gene Ther. 11:247. 24. Laouar, A., F. R. Collart, C. B. Chubb, B. Xie, and E. Huberman. 1999. Interaction between ␣51 integrin and secreted fibronectin is involved in macrophage differentiation of human HL-60 myeloid leukemia cells. J. Immunol. 162:407. 25. Aota, S., M. Nomizu, and K. M. Yamada. 1994. The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function. J. Biol. Chem. 269:24756. 26. Livant, D. L., R. K. Brabec, K. Kurachi, D. L. Allen, Y. Wu, R. Haaseth, P. Andrews, S. P. Ethier, and S. Markwart. 2000. The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice. J. Clin. Invest, 105:1537. 27. Livant, D. L., R. K. Brabec, K. J. Pienta, D. L. Allen, K. Kurachi, S. Markwart, and A. Upadhyaya. 2000. Anti-invasive, antitumorigenic, and antimetastatic activities of the PHSCN sequence in prostate carcinoma. Cancer Res. 60:309. 28. Clark, R. A. 1993. Basics of cutaneous wound repair. J. Dermatol. Surg. Oncol. 19:693. 29. Lohr, M., B. Trautmann, M. Gottler, S. Peters, I. Zauner, B. Maillet, and G. Kloppel. 1994. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins. Br. J. Cancer 69:144. 30. Arenberg, D. A., P. J. Polverini, S. L. Kunkel, A. Shanafelt, J. Hesselgesser, R. Horuk, and R. M. Strieter. 1997. The role of CXC chemokines in the regulation of angiogenesis in non-small cell lung cancer. J. Leukocyte Biol. 62:554. 31. Brooks, P. C., R. A. Clark, and D. A. Cheresh. 1994. Requirement of vascular integrin ␣ v 3 for angiogenesis. Science 264:569. 32. Friedlander, M., C. L. Theesfeld, M. Sugita, M. Fruttiger, M. A. Thomas, S. Chang, and D. A. Cheresh. 1996. Involvement of integrins ␣ v 3 and ␣ v 5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. USA 93:9764. 33. Kim, S., K. Bell, S. A. Mousa, and J. A. Varner. 2000. Regulation of angiogenesis in vivo by ligation of integrin ␣51 with the central cell-binding domain of fibronectin. Am. J. Pathol. 156:1345. 34. McCutcheon, J. C., S. P. Hart, M. Canning, K. Ross, M. J. Humphries, and I. Dransfield. 1998. Regulation of macrophage phagocytosis of apoptotic neutrophils by adhesion to fibronectin. J. Leukocyte Biol. 64:600. 35. Seljelid, R., S. Jozefowski, and B. Sveinbjornsson. 1999. Tumor stroma. Anticancer Res. 19:4809. 36. Sethi, T., R. C. Rintoul, M. Moore, A. C. MacKinnon, D. Salter, C. Choo, E. R. Chilvers, I. Dransfield, S. C. Donnelly, R. Strieter, and C. Haslett. 1999. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nat. Med. 5:662.
