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A Site on Laminin 5, AQARSAASKVKVSMKF, Induces Inflammatory Cell Production of Matrix Metalloproteinase-9 and Chemotaxis¹

Tracy L. Adair-Kirk^*, Jeffrey J. Atkinson^*, Thomas J. Broekelmann, Masayuki Doi, Karl Tryggvason, Jeffrey H. Miner^,, Robert P. Mecham^*, and Robert M. Senior^2,*,

Divisions of ^* Pulmonary and Critical Care Medicine and Renal Diseases, Department of Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine and Barnes-Jewish Hospital, St. Louis, MO 63110; and Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several peptide sequences in laminin 1, the -chain of laminin (Ln)-1, mediate biological responses in vitro, but Ln-1 is rare in vivo. Since Ln-5 and Ln-10, which contain the 3 and 5 chains, respectively, are the most prominent laminin heterotrimers in normal adult tissues and few functional domains in other laminin chains have been identified, we are investigating the 3 and 5 chains for biological activities. Incubation of mouse macrophages with the laminin 5 peptide AQARSAASKVKVSMKF resulted in marked increase in matrix metalloproteinase (MMP)-9 mRNA and gelatinolytic activity in the conditioned media, whereas the corresponding 3 peptide QQARDAANKVAIPMRF had no effect. AQARSAASKVKVSMKF also induced expression of MMP-14, while MMP-2, MMP-3, MMP-7, MMP-12, and MMP-13 were not induced by this peptide. Deletion analyses indicated that a minimal sequence of ASKVKVSMKF was sufficient for increasing MMP-9 expression. AQARSAASKVKVSMKF was also chemotactic for neutrophils and macrophages in vitro, and induced accumulation of neutrophils and macrophages in lung airspaces in vivo following intranasal instillation into mice. Comparable accumulation occurred in MMP-9-deficient mice, indicating that MMP-9 was not required for AQARSAASKVKVSMKF-induced inflammatory cell emigration in the lung. A scrambled version of the minimal peptide, KAKSFVMVSK, was inactive. These data indicate that laminin 5-derived peptides can induce inflammatory cell chemotaxis and metalloproteinase activity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inflammatory cells are recruited to sites of tissue injury by cell-derived and exogenous chemotactic factors and/or degraded extracellular matrix (ECM)³ components. Once at the site, inflammatory cells play a role in the extent of tissue remodeling, in part, by the regulated production of a family of matrix metalloproteinases (MMPs). MMPs are thought to be involved in proteolysis of ECM proteins such as laminins, exposing cryptic domains which can regulate cell adhesion, chemotaxis, and other cellular responses. MMPs are important molecules in the regulation of cell and tumor growth, wound healing, apoptosis, angiogenesis, tumor invasion, and the immune response (1). Dysregulation of MMP production has been implicated in diverse inflammatory diseases and neoplastic diseases.

Laminins are a family of integral basement membrane glycoproteins, each containing an -, -, and -chain that assemble into characteristic heterotrimeric structures (2, 3). The long arms of the three chains associate and form a helical coiled-coil region. The C-termini of -chains are unique in that they form a large globular structure termed the G-domain. To date, five , three , and three laminin chains have been identified, which assemble into at least fifteen laminin isoforms. The best-characterized laminin is Laminin (Ln)-1, which has an 1, 1, 1 chain composition (4). Ln-1 promotes cell adhesion, polarization, migration, and epithelial cell differentiation. Several sites on the Ln-1 molecule that mediate various biological responses have been identified at the peptide level (5, 6, 7, 8, 9, 10, 11, 12). It has been proposed that Ln-1 contains cryptic domains, potentially exposed during proteolysis, which elicit biological responses distinct from the intact Ln-1 molecule (9). Peptides containing the sequence SIKVAV located near the C-terminal globular domain of the laminin 1 chain induce expression of gelatinase B (gelB/MMP-9) by monocytes/macrophages (6, 8), whereas intact Ln-1 does not (9). The biologic importance of cryptic domains in the Ln-1 in vivo is unclear because laminin 1, the -chain of Ln-1, is not expressed in most adult tissues (13, 14, 15, 16, 17, 18). This suggests that the laminin in the adult matrix that regulates biological activities likely contains a different laminin -chain. However, few specific sites on other laminin chains have been identified to promote biological functions.

Immunohistochemical data indicate that Ln-5 (332) and Ln-10 (511) are the most prominent Ln heterotrimers in normal adult tissues (19, 20, 21, 22, 23). Therefore, we are investigating the 3 and 5 chains for biological activities. In the present work, we demonstrate that a synthetic laminin 5 chain peptide (AQARSAASKVKVSMKF), derived from the corresponding region of laminin 1 that contains the cryptic SIKVAV sequence, has the capacity to increase MMP-9 production by macrophages. In contrast, a peptide from the corresponding region of the laminin 3 chain (QQARDAANKVAIPMRF) does not. Furthermore, AQARSAASKVKVSMKF promotes MMP-9 release by neutrophils and is chemotactic for inflammatory cells in vitro and in vivo. These data indicate that laminin 5-derived peptides can mediate responses by inflammatory cells and suggests that laminins containing the 5 chain (Ln-10 and Ln-11) are likely laminins in the adult matrix that regulate these activities.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Synthetic laminin peptide

The synthetic peptide derived from the laminin 1 chain (YIGSR) and the intact Ln-1 isolated from the Engelbreth-Holm-Swarm tumor were obtained from Sigma-Aldrich (St. Louis, MO). Intact Ln-10 was isolated from HEK293 cells transfected with full-length cDNAs encoding the human laminin 5, 1, and 1 chains (24). The laminin-derived peptides were synthesized using standard FastMoc chemistry on an ABI431A peptide synthesizer. Peptides were purified using reverse phase (C18) HPLC and the sequence confirmed by mass spectrometry. The sequences of the laminin-derived peptides are as follows: 1 [aa 2096–2108 (25); SRARKQAASIKVAVSADR], 3 [aa 1613–1628 (26); QQARDAANKVAIPMRF], 5 [aa 2643–2658 (20); AQARSAASKVKVSMKF], 5f [AQARSAA], 5b [ASKVKVSMKF], 5b-sc [KAKSFVMVSK], 5b-pd1 [SKVKVSMKF], 5b-pd2 [AKVKVSMKF], and 5b-pd3 [ASVKVSMKF]. All peptides were dissolved in PBS at 5 mg/ml, filter sterilized, and stored at -20°C. Peptides were diluted to 25–200 µg/ml in macrophage serum-free media (MSFM) (Invitrogen, Carlsbad, CA) before incubation with cells.

Treatment of RAW264.7 macrophages with laminins or laminin-derived peptides

Murine RAW264.7 macrophages (American Type Tissue Culture, Rockville, MD) were maintained in low bicarbonate (1.5 g/L) DMEM, supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 4 mM L-glutamine (Invitrogen). Cells were suspended in complete medium and aliquoted into 24-well plates at 2.5 x 10⁵ cells/well. Following 3 h adherence, cells were washed 3 times with MSFM and then incubated with MSFM alone or MSFM containing intact laminin or laminin-derived peptides. At designated times, conditioned media were harvested, concentrated 5-fold using Amicon YM-30 Centricon centrifugal filter devices (Millipore, Bedford, MA), and assayed for MMP activity by zymography. In some experiments, cells were incubated in the presence of 5 µg/ml cycloheximide (Sigma-Aldrich) for 30 min before the addition of laminin peptides and for the duration of the experiment.

Zymography

Proteolytic activity in conditioned media was determined by zymography (27). Conditioned media were electrophoresed briefly in a 7.5% acrylamide gel containing 1 mg/ml gelatin, gels were washed in 2.5% Triton X-100 (Sigma-Aldrich), and incubated 12 h in buffer containing 50 mM Tris (pH 8.0), 10 mM CaCl₂, and 1 mM ZnCl₂. Following incubation, gels were stained with Coomassie blue, destained, and gelatinolytic activity was detected as clear bands on a blue background. For casein zymography, samples were electrophoresed in a 12% acrylamide gel containing 1 mg/ml casein and incubated 4 day at 37°C. Polymorphonuclear neutrophil (PMN) lysate containing MMP-9, or isolated MMP-12 (provided by S. D. Shapiro, Brigham and Women’s Hospital, Harvard University Medical School, Boston, MA), were run as positive controls for gelatin or casein zymography, respectively. Metalloproteinase activity was inhibited by incubating gels in 50 mM Tris (pH 8.0), 5 mM EDTA.

Quantitative densitometry

Zymography gels were negatively scanned and quantitative densitometry was performed using the NIH Image program. All quantification was done on exposures in which individual bands were not yet saturating.

RNA isolation and RT-PCR

The expression of MMPs by RAW264.7 cells was examined by RT-PCR. RNA was extracted from cells that were incubated with MSFM alone or MSFM containing 100 µg/ml of AQARSAASKVKVSMKF or 1 µM PMA (Sigma-Aldrich) by the ToTALLY RNA isolation kit (Ambion, Austin, TX). One µg of RNA was reverse transcribed using random hexamers using the GeneAmp RNA PCR Core kit (Roche Molecular Systems, Branchburg, NJ). Specific primers for MMP-2, MMP-3, MMP-9, MMP-12, MMP-13, MMP-14, and GAPDH were designed as previously described (28, 29, 30, 31). Oligonucleotides for MMP-7 (32) were provided by C. Wilson (Washington University School of Medicine, St. Louis, MO). Products of 690, 610, 650, 585, 665, 610, 550, and 690 bp were predicted for MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, MMP-14, and GAPDH, respectively. The resulting amplification products were electrophoresed on a 1% agarose gel and stained with ethidium bromide.

Animal treatment

Six to 8-wk-old 129 SvEv mice (Taconic Farm, Germantown, NY) were housed in a barrier animal facility under the veterinary care of the Department of Comparative Medicine at Washington University School of Medicine in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Mice were anesthetized by i.p. injection of 0.1 ml mouse mixture with each mouse receiving 87 mg/kg ketamine HCl (Fort Dodge Laboratories, Fort Dodge, IA) and 13 mg/kg xylazine HCl (Phoenix Pharmaceuticals, St. Joseph, MO). Euthanasia was accomplished by carbon dioxide narcosis.

Isolation of monocytes and neutrophils

After anesthesia, mice were injected i.p. with 0.1 ml of 1000 U/ml of heparin to prevent coagulation and blood was collected from the chest cavity following the severing of the dorsal aorta above the diaphragm. The heart was perfused with 1–2 ml PBS and the pooled fluid was layered on a discontinuous Histapaque (Sigma-Aldrich) gradient (27). Cells that sedimented at the interface were collected, washed several times with PBS, and the percentage of monocytes was assessed by Wright-stained cytospin (LeukoStat, Fisher Scientific, Pittsburgh, PA). Neutrophils, which pelleted during centrifugation of the Histapaque gradient, were washed twice with PBS and incubated with 2.5% dextran to remove monocytes and lymphocytes. Red blood cells were lysed with Tris-buffered ammonium chloride buffer (pH 7.2). The neutrophil purity was determined by Wright-stained cytospins.

Isolation of peritoneal macrophages and peritoneal neutrophils

To obtain resident peritoneal macrophages, 2 x 10 ml of saline was injected into the peritoneal space and the fluid was pooled. Thioglycollate-elicited peritoneal neutrophils were harvested from the peritoneal space with 2 x 10 ml saline 4 h after an i.p. injection of 1 ml of 4% thioglycollate (BD Biosciences, San Jose, CA). Following each cell preparation, cells were washed with saline and an aliquot was taken to assess the percentage of macrophages or neutrophils by Wright-stained cytospins.

Treatment of isolated monocytes, macrophages, and neutrophils with the laminin 5 peptide

Cell concentrations of isolated monocytes and peritoneal macrophages were adjusted to plate 2.5 x 10⁵ monocytes/macrophages per well in a 24-well plate in RPMI containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 4 mM L-glutamine (Invitrogen). Following 3 h of adherence, cells were washed three times with MSFM to remove the contaminating non-adherent lymphocytes. Cells were then incubated for 24 h with MSFM alone or 100 µg/ml laminin 5 peptide. Following incubation, the conditioned media were harvested and assayed for gelatinolytic activity by gelatin zymography. Isolated neutrophils (5 x 10⁵ cells/ml) were incubated in suspension in 15-ml polypropylene tubes in PBS alone or PBS containing 10^-5 M fMLP or 100 µg/ml (5 x 10^-5 M) laminin 5 peptide for various times. Following incubation, cells were removed and the solution was assayed for gelatinolytic activity by gelatin zymography.

Chemotaxis assay

Chemotaxis was assayed in modified Boyden chambers, as previously described (33). Briefly, intact laminin or laminin-derived peptides in HBSS with 1 mM CaCl₂, 1 mM MgCl₂, and 0.1% BSA were placed in the lower compartments of the chambers. Cell suspensions (1.5 x 10⁵ cells/well) of neutrophils or macrophages were placed in the upper compartments separated by polyvinylpyrolidone-free polycarbonate filters (Osmonics, Livermore, CA) with 3 µm or 5 µm pore size, respectively. Following 90 min incubation of the chamber at 37°C in 5% CO₂, the filter was Wright-stained, non-migrating cells were removed, and the number of migrating neutrophils or macrophages on the undersurface of the filter was counted in ten random high-power fields (x400) for each of the triplicate wells. HBSS alone in the lower compartments was used as a negative control. The cell counts were averaged and the standard errors calculated. Data represent at least three independent experiments.

Intranasal instillation of laminin-derived peptides

After anesthesia, 129 SvEv mice were placed supine and dropwise inoculated intranasally with 50 µl of PBS, LPS (200 µg) (Sigma-Aldrich), or laminin-derived peptide (200 µg). Following the inoculation, the mice were kept supine 1–2 min to ensure inhalation of the sample. At the appropriate time, the mice were killed by carbon dioxide narcosis and bronchoalveolar lavage (BAL) fluid was retrieved by injecting 3 x 1 ml of saline through the trachea and pooling the fractions. The total cell count of the BAL fluid was determined using a hemacytometer after lysing the RBC with Tris-buffered ammonium chloride solution (pH 7.2). Leukocyte differential cell counts were performed upon Wright-stained preparations. The BAL fluid was assayed for MMP activity by zymography. All experiments were performed at least three times using multiple mice per condition. Additional studies were performed using MMP-9-deficient (34) and MMP-2-deficient mice. MMP-2 heterozygous mice were kindly provided by S. Itohara (RIKEN Brain Science Institute, Wako, Japan) (35) and bred to homozygosity.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The linker region of the laminin chains is conserved

Synthetic laminin 1 peptides containing the SIKVAV sequence modulate tumor invasion, metastasis, angiogenesis, and proteinase production (6, 7, 8, 9, 10). This site is in the linker region between the coiled-coil region and the globular domain. The sequence of the linker regions of mouse laminin -chains is fairly conserved (Fig. 1), especially in the region encompassing the SIKVAV sequence. For example, the entire linker region of the laminin 5 chain is only 26.8% identical to the corresponding region of laminin 1, whereas the region immediately surrounding the SIKVAV peptide sequence is 73.3% identical between these laminin -chains. This sequence homology between -chain isoforms extends across species. This region of mouse laminin 5 is 96.4% and 89.3% identical to the corresponding regions of rat (36) and human (19) laminin 5 chains, respectively (not shown). Such a high degree of sequence similarity suggests that this region of the different -chain isoforms may have similar biological activities.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Comparison of the sequences of the "linker" regions of the mouse laminin -chains. A diagrammatic representation of Ln-1 is shown illustrating known active sites. The amino acid sequences of the "linker" region between the coiled-coil and the globular domains of the mouse laminin -chains are compared. Dots indicate aa in other laminin -chains that are identical to laminin 1 chain. Dashes are incorporated to maintain alignment.

Laminin 1- and 5-, but not 3-derived peptides up-regulate macrophage MMP-9 expression

Previous studies indicate that SIKVAV-containing peptides derived from the laminin 1 chain of Ln-1 can up-regulate macrophage MMP-9 expression (6, 8, 9). Here, we examined the ability of laminin 3 or 5 peptides derived from the corresponding region in laminin 1 to modulate MMP expression by macrophages. RAW264.7 macrophages were exposed to laminin-derived peptides for 24 h, and the gelatinase activity of the conditioned media was assayed by gelatin zymography. Incubation with the synthetic laminin 1 peptide SRARKQAASIKVAVSADR resulted in increased expression of a 105 kDa gelatinolytic band 6.3 fold, while intact Ln-1 had no effect (Fig. 2, B and C). This gelatinase comigrates with the MMP-9 in PMN lysates and exhibits the characteristic divalent cation-dependence of MMPs, as demonstrated by the loss of gelatinolytic activity during incubation of the gel in buffer containing EDTA (data not shown). These data confirm the findings of Khan and Falcone (8), who further demonstrated that the minimal essential sequence in this peptide is SIKVAV, with the Ile being critical. Despite the lack of a "critical" Ile, the laminin 5-derived peptide AQARSAASKVKVSMKF had 2.4 fold greater effect on the expression of MMP-9 compared with the laminin 1 peptide (Fig. 2C). The laminin 5 peptide’s effect on macrophage MMP-9 expression was dose- and time-dependent with MMP-9 activity in conditioned media visible at 6 h following exposure of RAW264.7 macrophages to laminin 5 peptide and increased over a 24 h period (data not shown). Furthermore, in the presence of cycloheximide, a protein synthesis inhibitor, AQARSAASKVKVSMKF failed to induce expression of MMP-9 by RAW264.7 macrophages. Taken together these data indicate that the laminin 5 peptide induces new MMP-9 synthesis.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Peptides derived from the -chains of laminins-1 (1) and -10 (5), but not -5 (3), up-regulate gelatinase expression by macrophages. A, RAW264.7 cells were aliquoted into 24-well plates at 2.5 x 105 cells/well. Following 3 h adherence, cells were incubated with either MSFM alone (Control) or MSFM containing 100 µg/ml of intact Ln-1, the laminin 1 peptide, intact Ln-10, the laminin 5 peptide, or the laminin 3 peptide. Following 24 h incubation, the conditioned media were assayed for gelatinase activity by gelatin zymography. B, The zymograms were negatively scanned, digitized, and quantified using the NIH Image program. The data represents the mean ± SEM from at least three independent experiments. The amino acid sequences of the synthetic laminin 1, 3, and 5 peptides are provided in Materials and Methods.

Laminin 5 peptide-induced macrophage MMP expression is restricted to MMPs-9 and -14

Macrophage matrix metalloproteinases include collagenase-1/MMP-1, gelatinase A/MMP-2, stromelysin-1/MMP-3, matrilysin/MMP-7, gelatinase B/MMP-9, macrophage elastase/MMP-12, and MT1-MMP/MMP-14 (37, 38, 39, 40, 41, 42, 43). A gene encoding MMP-1 has not been identified in mice; the proposed equivalent MMP is collagenase-3/MMP-13 (44). As shown above, the gelatinase up-regulated by macrophages by AQARSAASKVKVSMKF comigrated with MMP-9 at 105 kDa. Other gelatinases (e.g., MMP-2, 72 kDa; MMP-3, 57 kDa; MMP-13, 53kDa) were not detected. Furthermore, MMP-7 (28 kDa) or MMP-12 (22 kDa) were not detected by casein zymography (data not shown). These results suggest that the macrophage response to AQARSAASKVKVSMKF does not affect the expression of MMPs other than MMP-9. To further examine the induced expression of MMPs by the AQARSAASKVKVSMKF, RT-PCR analyses were performed using RNA isolated from RAW264.7 cells exposed to AQARSAASKVKVSMKF. RNA isolated from RAW264.7 cells treated with PMA was used as a positive control. The steady-state levels of MMP-2, MMP-3, MMP-7, or MMP-12 mRNAs did not increase in response to laminin 5 peptide (Fig. 3). In contrast, treatment with the laminin 5 peptide increased the levels of MMP-9 and MMP-14 mRNAs above the steady-state levels of mRNAs from the control cells. MMP-13 mRNA was undetectable even following incubation of RAW264.7 cells with PMA. These data indicate that AQARSAASKVKVSMKF-induced macrophage MMP expression is restricted, but the possibility that the laminin 5 peptide induces the expression of other proteases, such as serine proteases, cannot be ruled out.

View larger version (76K): [in this window] [in a new window]   FIGURE 3. Laminin 5 peptide-induced macrophage MMP expression is restricted to MMP-9 and MMP-14. RNAs isolated from RAW264.7 macrophages that were untreated (C), exposed to 100 µg/ml laminin 5 peptide for 24 h, or exposed to 1 µM PMA for 2 h were subjected to RT-PCR using primers specific for murine MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, MMP-14, or GAPDH. Resulting amplification products were electrophoresed in a 1% agarose gel and stained with ethidium bromide. PCR amplification conducted in absence of cDNA (-) was used as a negative control for each primer set.

Identification of amino acid in the laminin 5 peptide responsible for MMP-9 expression by macrophages

To determine the sequences in AQARSAASKVKVSMKF that elicit increased expression of MMP-9 by macrophages, we generated six synthetic laminin peptides related to AQARSAASKVKVSMKF. Treatment of RAW264.7 macrophages with a peptide derived from amino acid 2643 to 2649 of the laminin 5 chain, 5f, had no effect on the expression of MMP-9 (Fig. 4A). In contrast, treatment with a peptide derived from aa 2649 to 2658 of the laminin 5 chain, 5b, increased macrophage MMP-9 expression. To determine whether the response is sequence-specific, a synthetic peptide was generated which scrambled the sequence in 5b, called 5b-sc. This peptide, KAKSFVMVSK, had no effect on MMP-9 expression (Fig. 4A), indicating that ASKVKVSMKF interacts with the macrophage in a sequence-specific manner to induce MMP-9 expression. Interestingly, the YIGSR peptide, derived from the short arm of the laminin 1 chain that stimulates MMP-9 expression by tumor cells (7), also had no effect on macrophage MMP-9 expression. The region between aa 2649 and 2658 of the laminin 5 chain (5b, ASKVKVSMKF) corresponds to the region of laminin 1 containing the SIKVAV sequence. To further define the sequences in ASKVKVSMKF required for the up-regulation of macrophage MMP-9 expression, point deletions within the 5b peptide were generated. Singly deleting any of the first three aa in ASKVKVSMKF eliminated the peptide’s ability to up-regulate MMP-9 expression by RAW264.7 macrophages (Fig. 4B).

View larger version (76K): [in this window] [in a new window]   FIGURE 4. Identification of laminin 5 peptide sequence responsible for MMP-9 expression by macrophages. RAW264.7 cells were aliquoted into 24-well plates at 2.5 x 105 cells/well. Following 3 h adherence, cells were incubated with either MSFM alone (Control) or MSFM containing 100 µg/ml of the indicated peptides. Following 24 h incubation, the conditioned media were assayed for gelatinase activity by gelatin zymography. The amino acid sequences of the laminin peptides are provided in Materials and Methods.

Laminin 5 peptide up-regulates MMPs-9 and -14 by monocytes and macrophages

To determine whether the effect of AQARSAASKVKVSMKF on RAW264.7 macrophages extends to normal mouse monocytes and macrophages, these cell types were isolated and exposed to the laminin 5 peptide. Following a 24 h incubation with the laminin 5 peptide, an increase in MMP-9 activity was detected in the conditioned media from both monocytes and macrophages (Fig. 5A). Similar results were obtained using human blood monocytes (data not shown). RT-PCR analysis of RNA isolated from the laminin 5 peptide-treated cells revealed an increase in the levels of MMP-14 mRNA above the steady-state levels of mRNAs from the untreated control cells by macrophages, but not monocytes (Fig. 5B). Lack of MMP-14 expression by blood monocytes may not reflect an inability to respond to the laminin 5 peptide but rather an inability of undifferentiated monocytes to produce this proteinase.

View larger version (64K): [in this window] [in a new window]   FIGURE 5. Laminin 5 peptide induced MMP-9 and MMP-14 expression by isolated mouse macrophages. Blood monocytes and peritoneal macrophages were isolated from mice and plated at 2.5 x 105 cells/well into 24-well plates. Following 3 h adherence, non-adherent cells were removed and the remaining cells were incubated for 24 h with either serum-free media alone (C) or serum-free media containing 100 µg/ml laminin 5 peptide. Following incubation, the conditioned media were assayed for MMP-9 activity by gelatin zymography (A), and RNAs were isolated and subjected to RT-PCR using primers specific for murine MMP-14 (B). Resulting amplification products were electrophoresed in a 1% agarose gel and stained with ethidium bromide. Data from RAW264.7 macrophages are shown for comparison.

Laminin 5 peptide induces chemotactic migration of neutrophils and macrophages

Matrix components have been shown to influence other inflammatory cell functions (45, 46). Therefore, we examined whether the intact laminin molecules or the laminin-derived peptides have chemotactic activity for inflammatory cells. Chemotaxis assays were performed using modified Boyden chambers with resident peritoneal macrophages or thioglycollate-elicited peritoneal neutrophils. Similar to the effects on macrophage MMP-9 expression, both the laminin 1- and 5-derived peptides promoted macrophage and neutrophil migration in a dose dependent manner, whereas intact Ln-1 and Ln-10 did not (Figs. 6, A and B).

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Laminin 5 peptide is chemotactic for peritoneal macrophages and neutrophils. Chemotaxis assays were performed using modified Boyden chambers with resident peritoneal macrophages, (A), or thioglycollate-elicited peritoneal neutrophils, (B), Buffer alone (HBSS), or the indicated amount of intact Ln-1, laminin 1 peptide, intact Ln-10, or laminin 5 peptide was used as chemoattractant. The average number of macrophages/neutrophils per high-power field (HPF) ± SEM of triplicate samples is shown. Data are representative of at least three separate experiments.

Laminin 5 peptide induces MMP-9 release by neutrophils

Because many factors that promote neutrophil migration also stimulate neutrophil granule release, we tested the capacity of AQARSAASKVKVSMKF to cause release of MMP-9 that is stored in secondary granules. Treatment of neutrophils with the laminin 5 peptide caused the release of MMP-9 within 5 min of exposure (Fig. 7), plateauing by 30 min at a level comparable with that elicited by 60 min of treatment with the chemotactic peptide fMLP (47, 48).

View larger version (85K): [in this window] [in a new window]   FIGURE 7. Laminin 5 peptide induces MMP-9 release by neutrophils. Neutrophils were isolated from normal mice and incubated in suspension at 5 x 105 cells/ml in PBS alone (Control) or PBS with the laminin 5 peptide or fMLP. Following incubation, the cells were removed and the solution was assayed for gelatinolytic activity by gelatin zymography.

Laminin 5 peptide in vivo induces emigration of inflammatory cells and release of MMP-9 into alveolar spaces

To determine whether the laminin 5 peptide could induce emigration of neutrophils and macrophages in vivo, AQARSAASKVKVSMKF was instilled into the lungs of mice and the lungs were lavaged 1–3 days later. At 1 day postinstillation of the laminin 5 peptide, a marked increase in the number of alveolar neutrophils in the BAL fluid was observed as compared with the BAL fluid from PBS-treated mice (Fig. 8A). The potency of AQARSAASKVKVSMKF on inflammatory cell migration was similar to that of LPS, a factor known to elicit neutrophil accumulation in the lung (49). However, unlike LPS, the laminin 5 peptide also induced the migration of macrophages at later time points. Similar to its lack of induction of MMP-9 expression, the laminin 5f peptide AQARSAA had no effect on the emigration of neutrophils or macrophages, while the laminin 5b peptide ASKVKVSMKF that induced MMP-9 expression also induced emigration of neutrophils and macrophages. Furthermore, the scrambled 5b peptide, 5b-sc (KAKSFVMVSK), failed to induce emigration of these inflammatory cells. Similar results were obtained when the peptides were injected into the peritoneal cavity and peritoneal fluid analyzed for accumulation of inflammatory cells (data not shown).

View larger version (71K): [in this window] [in a new window]   FIGURE 8. Cell content and gelatinolytic activity in BAL fluid following intranasal administration of laminin 5 peptides. Mice were intranasally instilled with PBS alone or PBS containing 200 µg of 5, 5f, 5b, 5b-sc peptides, or LPS. The amino acid sequences of the laminin peptides are provided in Materials and Methods. One or 3 days postinstillation, BAL fluids were collected, and the cells were separated from the BAL fluids and characterized as indicated in Materials and Methods. (A),The number of neutrophils and macrophages per milliliter BAL fluid ± SEM is shown. Data are representative of three separate experiments using at least three mice per condition. BAL fluids collected at 1 (B) or 3 (C), days postinstillation were assayed for gelatinase activity by gelatin zymography.

At 3 days postinstillation, an increase in MMP-9 and MMP-2 gelatinolytic activities was observed in the BAL fluid from laminin 5 peptide-treated mice (Fig. 8C), which coincided with an increase in the number of macrophages (Fig. 8A). Our in vitro data (Fig. 3) indicates that the laminin 5 peptide does not directly induce MMP-2 expression by macrophages. Collectively, it appears that AQARSAASKVKVSMKF has effects on other cell types in vivo which causes the induction of MMP-2.

To determine whether emigration of macrophages and neutrophils by the laminin 5 peptide requires MMP-9, AQARSAASKVKVSMKF was intranasally instilled into the lungs of mice with a null mutation of the MMP-9 gene. The BAL fluid from MMP-9 null mice treated with the laminin 5 peptide had neutrophils and macrophages counts comparable to wild-type mice (data not shown). This is consistent with our earlier findings that neutrophil migration into tissues is unimpaired in MMP-9 deficient mice (30, 49).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Matrix components can influence inflammatory cell functions. For example, proteolytic fragments of ECM, such as fibronectin (50, 51), entactin, elastin (52, 53), and type I collagen (46), are chemotactic for inflammatory cells. In the present study, we investigated whether laminin molecules modulate inflammatory cell functions. Several sites on the Ln-1 molecule that mediate various biological responses have been identified at the peptide level (5, 6, 7, 8, 9, 10, 11, 12). Among these is a SIKVAV-containing peptide derived from the laminin 1 chain that modulates tumor invasion, metastasis, angiogenesis, and proteinase production (6, 7, 8, 9, 10). Interestingly, SIKVAV is in the linker region between the coiled-coil region and the globular domain of the laminin 1 chain, and this region is fairly conserved among the laminin -chains, suggesting that this region of the different laminin isoforms can serve similar biological functions. In these studies, we used synthetic peptides derived from the corresponding region of the laminin 3 and 5 chains, the -chains of Ln-5 and Ln-10, respectively, because these laminins are the most prominent Ln heterotrimers in normal adult tissues (19, 20, 21, 22, 23). We found that this region in the 1 and 5 chains induces inflammatory cell production of MMP-9 and migration in vitro and in vivo. In contrast, this region in the 3 chain fails to induce MMP-9 expression by macrophages. These data establish the specificity of the response and confirm that not all laminin -chain linker region sequences are biologically active.

The biologic importance of Ln-1, or proteolytic fragments of it, in inflammatory cell protease production and chemotaxis in vivo is unknown, because Ln-1 is not expressed in most adult tissues (13, 14, 15, 16, 17, 18). Our data indicate that laminin-5 derived peptides mediate responses by inflammatory cells, including protease production and chemotaxis. Taken together with the wide tissue distribution of the laminin 5 chain in both adult and fetal tissues (19, 20, 21, 22, 23), our data suggest that laminins containing the 5 chain are likely laminins in the adult matrix which affect these activities. Nonetheless, we cannot rule out the possibility that the expression of Ln-1 may increase during injury situations. Ln-1 fragments have been detected in extracts of abdominal aortic aneurysm using a polyclonal Ab against the 1 chain of Ln-1 (54). However, due to the amino acid conservation between laminin -chains, it is possible that this Ab also recognizes other laminin -chains so that the activity seen could be due to other more prominent laminin heterotrimers.

Interactions between inflammatory cells and the ECM are mediated via cell surface receptors, including members of the integrin family. Ln-1 has been shown to bind to several integrins (₁₁, ₂₁, ₃₁, ₅₁, ₆₁, ₇₁, ₆₄, and _V3) (16, 55, 56, 57), while Ln-10 only binds to a subset of these integrins (₃₁, ₆₁, ₆₄, and ₇₁) (57, 58). Inflammatory cells express many integrin receptors, including ₁₁, ₂₁, ₃₁, ₅₁, ₆₁, and _V3 (59, 60). Although the identity of the receptor remains to be determined, preliminary data suggest that the laminin 1 and 5 peptides use the same receptor for chemotaxis (data not shown).

The ability of laminins to modulate inflammatory cell functions may require the exposure of cryptic domains. Our research, as well as others, has demonstrated that the synthetic SIKVAV-containing laminin 1 peptide up-regulates macrophage MMP-9 expression, whereas intact Ln-1 has no effect (9). Furthermore, we observed that intact Ln-1 and Ln-10 fail to stimulate the migration of neutrophils or macrophages, while the SIKVAV-containing laminin 1 peptide and the laminin 5 peptide, ASKVKVSMKF, do modulate inflammatory cell migration in vitro. These findings suggest that laminin peptides, which may be generated or exposed during proteolytic digestion of laminins, can stimulate macrophage and neutrophil migration. This data is the first to show that a region of any laminin -chain has chemotactic activity. Whether the laminin 5 peptide acts directly or indirectly to promote inflammatory cell migration in vivo is unclear. Although the laminin 1 and 5 peptides have direct effects on neutrophil and macrophage migration in vitro, their effectiveness is less than known chemoattractants, such as fMLP (data not shown). However, when introduced into the lung, ASKVKVSMKF promotes macrophage and neutrophil emigration into alveolar spaces at comparable levels to LPS. Accordingly, the in vivo effects of the laminin 5 peptide may be more complex, possibly involving other cell types that amplify the chemotactic response.

The physiological role(s) of MMP-9 production by inflammatory cells is not known. Since some basement membrane components are substrates for MMP-9, it has been speculated that secretion of MMP-9 from these cells would facilitate their emigration. This hypothesis has been supported by studies showing that human neutrophil chemotaxis through an artificial basement membrane is impeded by blocking MMP-9 activity (61). However, in the present study we found that MMP-9 deficient neutrophils and macrophages migrated normally into alveolar spaces in response to the laminin 5 peptide. This finding supports earlier data in which neutrophil migration into tissues is unimpaired in MMP-9 deficient mice (30, 49). Therefore, our data show that production of MMP-9 and chemotactic migration are distinct and separate responses to the laminin 5 peptide.

Repair of injured epithelium is necessarily accompanied by clearing of the provisional matrix allowing its replacement by normal basement membranes composed principally of type IV collagen, laminins, entactin, and perlecan. By cleaving ECM components, it is hypothesized that MMPs play a role in the repair of damaged epithelium (62, 63). In contrast, excessive or aberrant expression of MMPs has also been implicated in various destructive diseases, including arthritis, aortic aneurysms, multiple sclerosis, cancer metastasis, and several lung diseases, including asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome (64, 65). Our data suggests that laminin-derived fragments play a role in the emigration of neutrophils and macrophages into sites of injury, as well as increased production of MMP-9. Whether these activities affect tissue remodeling and repair or further contribute to the pathophysiology of chronic inflammatory diseases remains to be determined.

In conclusion, we found that the AQARSAASKVKVSMKF sequence in the laminin 5 chain of Ln-10 and Ln-11 has the capacity to increase MMP-9 and MMP-14 production by macrophages, promote MMP-9 release by neutrophils, and induce macrophage and neutrophil chemotactic migration both in vitro and in vivo. The ability of laminin peptides to modulate the production of MMP-9 by macrophages and neutrophils and recruitment of these inflammatory cells suggests that this domain in Ln-10 may play a role in the inflammatory response at sites of tissue injury and repair.

	Acknowledgments

 
We thank Gail Griffin and Diane Kelley for technical support, and Richard Pierce and J. Michael Shipley for advice and critical evaluation of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health, National Heart, Lung, and Blood Institute, HL29594 and HL47328, the Alan A. and Edith L. Wolff Charitable Trust (to R.M.S.), and National Institutes of Health, 5T32 HL07317-24 (to T.L.A.K.), and a Research Fellowship from the American Lung Association (to J.J.A.), and grants from the Novo Nordisc Foundation, the Swedish Medical Research Council, and European Community Contract QLK3-CT2000-00084 (to K.T.).

² Address correspondence and reprint requests to Dr. Robert M. Senior, Department of Medicine, Barnes-Jewish Hospital, North Campus, 216 South Kingshighway Boulevard, St. Louis, MO 63110. E-mail address: seniorr{at}msnotes.wustl.edu

³ Abbreviations used in this paper: ECM, extracellular matrix; Ln, laminin; MMP, matrix metalloproteinase; MSFM, macrophage serum-free media; PMN, polymorphonuclear neutrophil; BAL, bronchoalveolar lavage.

Received for publication July 30, 2002. Accepted for publication April 24, 2003.

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