©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Laminin SIKVAV Peptide Induction of Monocyte/Macrophage Prostaglandin E and Matrix Metalloproteinases (*)

Marta L. Corcoran (1)(§), Maura C. Kibbey (2), Hynda K. Kleinman (2), Larry M. Wahl (1)(¶)

From the (1) Cellular Immunology Section, Laboratory of Immunology and the (2) Cell Biology Section, Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892-4352

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The laminin-derived synthetic peptide containing the SIKVAV (Ser-Ile-Lys-Val-Ala-Val) amino acid sequence has been previously shown to regulate tumor invasion, metastasis, and angiogenesis. Here, we demonstate that this peptide also modulates human monocyte responses. Moreover, the monocytic responses elicited by this peptide are influenced by the culture conditions. When elutriated monocytes were cultured on SIKVAV substrate or in suspension with this peptide, the synthesis of prostaglandin E, interstitial collagenase, and gelatinase B was induced and was further enhanced in the presence of concanavalin A (ConA). However, when monocytes were adhered before adding soluble SIKVAV, the peptide alone failed to induce the production of prostaglandin E or matrix metalloproteinases. If adherent monocytes were exposed to SIKVAV in the presence of ConA, this peptide enhanced the ConA induced production of these mediators. In contrast to SIKVAV, the intact laminin molecule failed to influence these monocyte responses. This is the first demonstration that a laminin derived peptide is capable of inducing or enhancing monocyte inflammatory responses that may influence a number of biological activities such as wound healing or excessive connective tissue destruction associated with chronic inflammation.


INTRODUCTION

Monocytes are recruited to sites of tissue injury or chronic inflammation by cell-derived chemotactic factors and/or degraded extracellular matrix components. Once at the site, the monocyte may orchestrate the extent of connective tissue destruction, in part, by the production and activation of a family of metalloproteinases that degrade all the major components of the extracellular matrix (for review, see Birkedal-Hansen et al. (1993)). In addition to these matrix metalloproteinases (MMPs),() monocytes secrete inflammatory mediators such as prostaglandin E (PGE), tumor necrosis factor, and interleukin-1 (IL-1) which influence the production of MMPs by other cells. Treatment with lipopolysaccharide or concanavalin A (ConA) induces monocytes to produce PGE which leads to the production of MMPs (Wahl and Lampel, 1987). Conversely, agents that suppress monocyte production of PGE, such as anti-inflammatory drugs (Wahl and Lampel, 1987), interferon- (Wahl et al., 1990), IL-4 (Corcoran et al., 1992), and IL-10 (Mertz et al., 1994), also inhibit the production of MMPs. Thus, the synthesis of MMPs by monocytes is dependent on PGE production.

Matrix components have also been shown to influence monocyte functions. Exposure of monocytes to type I collagen, gelatin, and endothelial cell-derived basement membrane induces the production of PGE (Gudewicz et al., 1994). Native and denatured type I collagen also induce the production of monocyte interstitial collagenase (Shapiro et al., 1993) as well as a number of responses by alveolar macrophages including chemotaxis, oxidative burst, cytotoxicity, and production of elastase and gelatinase (Laskin et al., 1994). Although intact laminin does not induce the production of MMPs by macrophages/monocytes (Shapiro et al., 1993), we examined whether specific sites on laminin, possibly exposed during degradation of this molecule, could modulate monocyte function.

The glycoprotein laminin-1, a major component of the basement membrane, is composed of three chains (1, 1, and 1) which form a covalent cross-like molecule (Burgson et al., 1994). Laminin promotes cell adhesion, migration, differentiation, and tumor metastasis. Several sites on the laminin molecule that mediate these biological responses have been identified at the synthetic peptide level (Kleinman et al., 1993). One of these, the SIKVAV site (Ser-Ile-Lys-Val-Ala-Val), located on the long arm of the laminin 1 chain has been shown to promote attachment, migration, angiogenesis (Kibbey et al., 1994), protease production, tumor growth, and metastasis (Kanemoto et al., 1991; Tashiro et al., 1991; Grant et al., 1992; Kibbey et al., 1992; Sweeney et al., 1991; Stack et al., 1991). Thus, this peptide has the potential to regulate a number of cellular responses in a cell type-specific manner.

We demonstrate in this report that the laminin peptide SIKVAV increases the production of PGE, interstitial collagenase (EC 3.4.24.7) and gelatinase B (EC 3.4.24.35) by monocytes. These data suggest that laminin fragments may play an important role in the resolution of tissue damage at a wound site and/or the exacerbation of connective tissue destruction associated with chronic inflammatory lesions.


MATERIALS AND METHODS

Monocyte Purification

Peripheral blood mononuclear cells were obtained by leukapheresis of normal volunteers at the Department of Transfusion Medicine at the National Institutes of Health. The monocytes in the mononuclear fraction were enriched to 90-95% by counterflow centrifugal elutriation using endotoxin-free reagents as described previously (Wahl et al., 1984).

Laminin Peptide Production and Purification

The 1 chain-derived SIKVAV-containing peptide (amino acids 2099-2105; CRKQAASIKVAVS) was synthesized, purified as described previously (Graf et al., 1987) and endotoxin-filtered. Control peptides include the SHA-7 peptide (CSRARKQAASGKVAVSADR) which contains a single amino acid substitution in the SIKVAV active site, and YOSH-3 (CRTDEGEKKCGCPGC) which is an inactive sequence from the 1 chain of laminin. These peptides tested negative for endotoxin by the limulus amebocyte lysate assay.

Culture Conditions

Monocytes (20 10/4 ml) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) for 48 h at 37 °C under the following conditions: in suspension in 15-ml polypropylene tubes (Falcon) along with the peptides, in 60-mm tissue culture dishes that had been previously coated with peptides, or adhered to 60-mm culture dishes prior to the addition of peptides. Peptides were coated onto 60-mm tissue culture dishes by drying sterile phosphate-buffered saline containing the laminin peptides. Phosphate-buffered saline that did not contain peptide was used as the control.

Monocyte PGE and Matrix Metalloproteinase Assays

After 48 h of incubation under the culture conditions indicated above, the conditioned media were harvested and the levels of PGE in the supernatants were determined by radioimmunoassay (Wahl, 1981). Western blot analysis for the MMPs was performed as described previously (Corcoran et al., 1992). Briefly, the secreted proteins in the remaining supernatants were ethanol-precipitated, separated on reducing 8-16% Tris/glycine polyacrylamide gels and transferred onto a nitrocellulose membrane. A 1:100 dilution of antibody 110, which recognizes the latent and active forms of gelatinase B (92-kDa type IV collagenase), and antibody 125, which recognizes the active forms of interstitial collagenase was used followed by an incubation with 1:16,000 dilution of protein G-horseradish peroxidase. The MMP proteins were visualized by chemoluminescence (ECL kit, Amersham Corp.) and the intensity of the signal quantitated using Image 1.45 software and an Arcus scanner.


RESULTS

Effect of Substrate-bound SIKVAV on Monocyte/Macrophage Function

Freshly isolated human monocytes were added to tissue culture dishes that were previously coated with the laminin peptides. After 48 h, the conditioned media were evaluated for the presence of inflammatory mediators including interstitial collagenase, gelatinase B, and PGE. As determined by Western blot analysis, monocytes cultured on either 100 µg or 200 µg/well of SIKVAV-containing peptide induced the production of interstitial collagenase which was predominantly the 47- and 42-kDa catalytically active species (Fig. 1). Induction of interstitial collagenase was not observed when monocytes were cultured on YOSH-3, an inactive peptide sequence from the 1 chain of laminin, or on uncoated plastic. Native laminin did not induce the production of these mediators (data not shown). As expected, 20 µg/ml of ConA stimulated production of interstitial collagenase on nontreated dishes. Adding 20 µg/ml of ConA to the monocytes cultured on SIKVAV-coated dishes further enhanced the production of interstitial collagenase over that produced by monocytes cultured in the presence of SIKVAV alone (Fig. 1).


Figure 1: Effect of substrate bound laminin SIKVAV peptide on monocyte interstitial collagenase production. Elutriated monocytes (20 10/4 ml) were added to 60 mm tissue culture wells that were previously coated with no peptide or 100-200 µg of either SIKVAV or YOSH-3 per well. The monocytes were incubated in the presence or absence of 20 µg/ml of ConA for 48 h. The proteins in the conditioned media were ethanol precipitated and analyzed by Western blot with an antibody against interstitial collagenase as described in the Materials and Methods. This Western shows both the 42 and 47-kDa catalytically active collagenase protein.



Monocytes cultured on SIKVAV-coated dishes also produced gelatinase B (Fig. 2). A control peptide, SHA-7, which contains a single amino acid substitution (SGKVAV) in the biologically active site, did not induce the production of this enzyme. These results were also confirmed by gelatin zymography (data not shown). Since PGE production correlates with the production of monocyte MMPs (Corcoran et al., 1992), the levels of this mediator were also determined. The production of PGE (4.03 ± 1.4 ng/ml) by monocytes cultured on substrate-bound SIKVAV peptide (100 µg/ml) was 4-fold higher compared with no peptide treatment (1 ± 0.4 ng/ml). Addition of 1 µg/ml of ConA to the SIKVAV-coated dishes induced more than a 2-fold increase in PGE production (22.5 ± 2.4 ng/ml) over that observed from cells cultured on plastic and stimulated with ConA alone (9.9 ± 1.7 ng/ml).


Figure 2: Effect of substrate bound laminin SIKVAV peptide on monocyte production of latent gelatinase B. Human monocytes (20 10/4 ml) were added to 60 mm wells that had been previously treated with no peptide or 200-400 µg of SIKVAV peptide per well. The proteins secreted after a 48 h incubation were ethanol precipitated and Western blotted with an antibody against 92-kDa gelatinase B as described in the Materials and Methods.



Effect of Soluble SIKVAV on Adhered Monocytes

Monocytes cultured on tissue culture plates prior to addition of soluble SIKVAV peptide did not produce interstitial collagenase (Fig. 3). However, if the adhered monocytes were treated with 1 µg/ml ConA as well as soluble SIKVAV (400 µg/ml), the production of interstitial collagenase was significantly enhanced over that observed with ConA (1 µg/ml) treatment alone (Fig. 3). Similarly, the addition of SIKVAV peptide alone to adherent monocytes did not induce PGE production. However, the PGE produced in response to 1 µg/ml of ConA (6.5 ± 1.9 ng/ml versus control, 0.85 ± 0.2 ng/ml) was enhanced 4-5-fold (29 ± 7 ng/ml) by 100 µg/ml soluble SIKVAV peptide.


Figure 3: Modulation of interstitial collagenase production by adherent monocytes with soluble laminin SIKVAV peptide. Freshly isolated monocytes (20 10/4 ml) were adhered to 60 mm tissue culture dishes for 30 min prior to addition of soluble peptides. After a 48 h incubation period, the proteins in the supernatants were ethanol precipitated and Western blotted with an antibody against interstitial collagenase as described in Materials and Methods.



Effect of Soluble SIKVAV Added to Monocytes in Suspension

When the monocytes were cultured with the peptides in suspension, SIKVAV alone was also shown to induce interstitial collagenase in a dose-dependent manner (Fig. 4). While soluble SIKVAV alone (200 or 400 µg/ml) induced the production of interstitial collagenase, when a suboptimal stimulatory dose of ConA (1 µg/ml) was added with SIKVAV there was a synergistic induction of interstitial collagenase (Fig. 4). There was a positive correlation between MMP and PGE production. Suspended monocytes treated with soluble SIKVAV (100 µg/ml) peptide had a 5-fold increase in PGE levels (8.5 ± 0.1 ng/ml) as compared with untreated cells (1.6 ± 0.9 ng/ml).


Figure 4: Effect of soluble laminin SIKVAV peptide on interstitial collagenase production by monocytes cultured in suspension. SIKVAV laminin peptide was added to monocytes (20 10/4 ml) in suspension and cultured for 48 h in the absence or presence of ConA (1 µg/ml). Stimulation with 20 µg/ml of ConA is also shown as a positive control. The conditioned media were ethanol precipitated and Western blotted with an antibody against interstitial collagenase as described in the Materials and Methods.



Soluble SIKVAV (100 µg/ml) peptide also elicited the production of latent gelatinase B by monocytes cultured in suspension (Fig. 5). In the presence of ConA (20 µg/ml), SIKVAV enhanced by 4-5-fold the production of the 92-kDa gelatinase B over that induced with ConA treatment alone (Fig. 5). Additionally, the 84-kDa proteolytically active product of gelatinase B was detected when monocytes were treated with ConA and SIKVAV.


Figure 5: Effect of soluble laminin SIKVAV peptide on the production of latent and active gelatinase B by monocytes cultured in suspension. Human monocytes (20 10/4 ml) were cultured in suspension for 48 h in the presence or absence of SIKVAV peptide and ConA (20 µg/ml) as indicated. The proteins in the media were ethanol precipitated and Western blotted using an antibody against gelatinase B as described in the Materials and Methods. This Western shows both the latent 92-kDa species as well as the catalytically active 84-kDa product.




DISCUSSION

Monocytes were cultured under different conditions to simulate situations that occur either in the blood stream or at sites of tissue damage. When the monocytes were cultured in suspension, SIKVAV stimulated the production of PGE, interstitial collagenase, and gelatinase B in the absence of a primary stimulus (ConA). In the presence of suboptimal concentrations of ConA, monocytes cultured in suspension responded to SIKVAV by inducing a greater enhancement of monocyte PGE and MMPs. Substrate-bound SIKVAV also directly elicited the production of PGE, interstitial collagenase and gelatinase B, and enhanced these responses in the presence of a suboptimal concentration of ConA. However, when monocytes were adhered, SIKVAV failed to induce MMP and PGE production, but enhanced these responses when they were initiated by ConA. The fact that SIKVAV can directly induce monocyte protease production agrees with previous studies on endothelial cells (Grant et al., 1992). The differential responses to SIKVAV observed under different culture conditions may be important in understanding the control mechanisms that limit monocyte activation at sites of inflammation and tissue damage.

Cellular invasion studies using the U937 monoblastic cell line suggest that gelatinase B may be the protease utilized by monocytes to transverse the extracellular matrix and enter inflammatory sites (Watanabe et al., 1993). Once at the inflammatory site, the production of interstitial collagenase by these cells is pivotal for initiating the degradation of fibrillar collagen in the damaged matrix. Stimulation of monocyte interstitial collagenase, gelatinase B, PGE, and possibly other monocyte functions by SIKVAV may be important in promoting tissue repair during wound healing.

The ability of degraded extracellular matrix molecules to elicit an inflammatory response by human monocytes, such as the production of tumor necrosis factor, has been observed with fibronectin and its fragments (Beezhold and Personius, 1992; Chang et al., 1993). Other studies have reported that the manner in which the monocytes are cultured may modulate their responses to extracellular matrix molecules (Kohn and Klingemann, 1991). Native and denatured collagens have also been reported to induce the production of arachidonic acid metabolites by monocytes (Gudewicz et al., 1994) and interstitial collagenase by alveolar macrophages (Shapiro et al., 1993). Collagen and collagen fragments also enhance chemotaxis, respiratory burst, gelatinase, and elastase production by alveolar macrophages (Laskin et al., 1994). Although native laminin has been previously observed to be inert in stimulating monocyte function (Shapiro et al., 1993; Gudewicz et al., 1994), we show here that the laminin-derived amino acid sequence SIKVAV, by itself, induces an inflammatory response from human monocytes. This suggests that this site of laminin contributes not only to promote angiogenesis but also to potentiate monocytic inflammatory responses.

Previously, SIKVAV has been reported to promote angiogenesis by inducing endothelial cell adhesion, migration, and invasion (Kibbey et al., 1992). In a murine model, injection of matrigel along with the SIKVAV peptide results in vascularization of the matrigel plug. This effect may be partly due to the infiltration of polymorphonuclear cells (PMNs) and subsequently monocytes (Kibbey et al., 1994) into the SIKVAV-containing matrigel plug. If these mice are made neutropenic, the angiogenesis in the matrigel plug containing SIKVAV is greatly reduced. Moreover, we also demonstrated that PMNs have the capacity to release proteases that degrade intact laminin molecules. It is possible that the PMNs, one of the first cells to arrive at the inflammatory site, initiate the degradation of matrix components such as collagen and laminin. The degradation of laminin and subsequent exposure of peptides containing the SIKVAV sequence may potentiate the inflammatory response by infiltrating monocytes. The ability of laminin peptides to modulate the production of PGE, interstitial collagenase, as well as the gelatinase B by monocytes could play an important role in their ability to transverse basement membranes. Thus, laminin fragments containing the SIKVAV sequence may contribute to the inflammatory response at sites of wound healing. Additionally, laminin fragments containing the SIKVAV sequence may contribute to the excessive induction of metalloproteinases that ultimately result in destruction of connective tissue associated with chronic inflammatory lesions.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: National Institutes of Health, National Cancer Institute, Laboratory of Pathology, Extracellular Matrix Pathology Section, Bldg. 10, Rm. 2A33, Bethesda, MD 20892-4352.

To whom correspondence should be addressed: 30 Convent Dr., MSC 4352, NIDR, National Institutes of Health, Rm. 325, Bethesda, MD 20892-4352. Tel.: 301-496-9219; Fax: 301-402-1064.

The abbreviations used are: MMPs, matrix metalloproteinases; PGE, prostaglandin E; ConA, concanavalin A; IL-1, interleukin-1; PMNs, polymorphonuclear cells.


ACKNOWLEDGEMENTS

We thank Dr. William Stetler-Stevenson of the Extracellular Matrix Pathology, Laboratory of Pathology, National Cancer Institute for providing the antibodies against interstitial collagenase and gelatinase B and Susan Hopkinson for her excellent technical assistance.


REFERENCES
  1. Beezhold, D. H., and Personius, C.. (1992) J. Leukocyte Biol. 51, 59-64 [Abstract]
  2. Birkedal-Hansen, H., Moore, W. G. I., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., and Engler, J. A. (1993) Crit. Rev. Oral Biol. Med. 4, 197-250 [Abstract]
  3. Burgson, R. E., Chiquet, M., Duetzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvason, K., Yamada, Y., and Yurchenco, P. D. (1994) Matrix Biol. 14, 209-211 [CrossRef][Medline] [Order article via Infotrieve]
  4. Chang, Z. L., Beezhold, D. H., Personius, C. D., and Shen, Z. L. (1993) J. Leukocyte Biol. 53, 79-85 [Abstract]
  5. Corcoran, M. L., Stetler-Stevenson, W. G., Brown, P. D., and Wahl, L. M. (1992) J. Biol. Chem. 267, 515-519 [Abstract/Free Full Text]
  6. Graf, J., Ogle, R. C., Robey, F. A., Sasaki, M., Martin, G. R., Yamada, Y., and Kleinman, H. K. (1987) Biochemistry 26, 6896-6904 [Medline] [Order article via Infotrieve]
  7. Grant, D. S., Kinsella, J. L., Fridman, R.. Auerbach, R., Piasecki, B. A., Yamada, Y., Zain, M., and Kleinman, H. K. (1992) J. Cell. Physiol. 153, 614-625 [Medline] [Order article via Infotrieve]
  8. Gudewicz, P. W., Frewin, M. B., Heinel, L. A., and Minnear, F. L. (1994) J. Leukocyte Biol. 55, 423-429 [Abstract]
  9. Kanemoto, T., Martin, G. R., Hamilton, T. C., and Fridman, R. (1991) Invasion & Metastasis 11, 84-92
  10. Kibbey, M. C., Grant, D. S., and Kleinman, H. K. (1992) J. Natl. Cancer Inst. 84, 1633-1638 [Abstract]
  11. Kibbey, M. C., Corcoran, M. L., Wahl, L. M., and Kleinman, H. K. (1994) J. Cell. Physiol. 160, 185-193 [Medline] [Order article via Infotrieve]
  12. Kleinman, H. K., Weeks, B. S., Schnaper, H. W., Kibbey, M. C., Yamamura, K., and Grant, D. S. (1993) Vitam. Horm. 47, 161-86 [Medline] [Order article via Infotrieve]
  13. Kohn, F. R., and Klingemann, H. G. (1991) Exp. Hematol. 19, 653-658 [Medline] [Order article via Infotrieve]
  14. Laskin, D. L., Soltys, R. A., Berg, R. A., and Riley, D. J. (1994) Am. J. Respir. Cell. Mol. Biol. 10, 58-64 [Abstract]
  15. Mertz, P. M., DeWitt, D. L., Stetler-Stevenson, W. G., and Wahl, L. M. (1994). J. Biol. Chem. 269, 21322-21329 [Abstract/Free Full Text]
  16. Shapiro, S. D., Kobayashi, D. K., Pentland, A. P., and Welgus, H. G. (1993) J. Biol. Chem. 268, 8170-8155 [Abstract/Free Full Text]
  17. Stack, S., Gray, R. D., and Pizzo, S. V. (1991) Biochemistry 30, 2073-2077 [Medline] [Order article via Infotrieve]
  18. Sweeney, T. M., Kibbey, M. C., Zain, M., Fridman, R., and Kleinman, H. K. (1991) Cancer Metastasis Rev. 10, 245-254 [Medline] [Order article via Infotrieve]
  19. Tashiro, K., Sephel, G. C., Greatorex, D., Sasaki, M., Shirashi, N., Martin, G. R., Kleinman, H. K., and Yamada, Y. (1991) J. Cell. Physiol. 146, 451-459 [Medline] [Order article via Infotrieve]
  20. Wahl, L. M. (1981) in Manual of Macrophage Methodology (Herscowitz, H. B., Holden, H. T., Bellanti, J. A., and Ghaffar, A., eds) pp. 423-429, Marcel Dekker, Inc., New York
  21. Wahl, L. M., and Lampel, L. L. (1987) Cell Immunol. 105, 411-422 [Medline] [Order article via Infotrieve]
  22. Wahl, L. M., Katona, M. I., Wilder, R. L., Winter, C. C., Haraoui, B., Sher, I., and Wahl, S. M. (1984) Cell Immunol. 85, 373-383 [Medline] [Order article via Infotrieve]
  23. Wahl, L. M., Corcoran, M. L., Mergenhagen, S. E., and Finbloom, D. S. (1990) J. Immunol. 144, 3518-3522 [Abstract/Free Full Text]
  24. Watanabe, H., Nakanishi, I., Yamashita, K., Hayakawa, T., and Okada, Y. (1993) J. Cell Sci. 104, 991-999 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.