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. 2007 Mar 21;2(3):e312.
doi: 10.1371/journal.pone.0000312.

LPS responsiveness and neutrophil chemotaxis in vivo require PMN MMP-8 activity

Angus M Tester  1 , Jennifer H Cox, Andrea R Connor, Amanda E Starr, Richard A Dean, Xose S Puente, Carlos López-Otín, Christopher M Overall
Affiliations

LPS responsiveness and neutrophil chemotaxis in vivo require PMN MMP-8 activity

Angus M Tester et al. PLoS One. .

Abstract

We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser(4)-Val(5) and Lys(79)-Arg(80). LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4 approximately 5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg(5)-Ser(6) and at Val(7)-Leu(8) in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Impaired PMN responsiveness to LPS in MMP-8 deficient mice.
(A) Infiltration of PMNs in vivo in response 1 µg of LPS (n = 8) or phosphate buffered saline control (n = 4) injected into the air pouch of male Mmp8-/- (black bar) and wild type mice (white bar) was assessed 8 h post-injection. The PMN influx was quantitated by myeloperoxidase activity. Error bars, standard error. (B) Western blot analysis of murine MMP-8 in LPS-treated air pouch PMN lysates corresponding to 50,000 cells per lane.
Figure 2
Figure 2. MMP-8 Cleavage of LIX.
(A) Tris-tricine 15% SDS-PAGE gel analysis of MMP-8 cleavage of murine CXCR2-binding chemokines DCIP-1, KC, LIX and MIP-2. The m/z [M+H]+ of reaction products are as shown. (B) Identification of cleavage products by MALDI-TOF mass spectrometry and NH2-terminal Edman sequencing. n.d., not determined. (C) Tris-tricine 15% SDS-PAGE and MALDI-TOF analysis of LIX cleavage products generated over time (h) by MMP-8. (D) Tris-tricine 15% SDS-PAGE and MALDI-TOF analysis of LIX cleavage by MMP-8 in the presence of 10-fold molar excess of recombinant hemopexin C-domain (MMP-8 CD), 10 µM EDTA, or 10 µM BB94. (E) Heparin Sepharose AKTA chromatograms of synthetic analogues of MMP-8 cleavage products of LIX eluted with a linear gradient of NaCl as indicated.
Figure 3
Figure 3. LIX is selectively cleaved by multiple MMPs.
(A) Tris-tricine SDS-PAGE and MALDI-TOF mass spectrometry analysis of MMP processing of LIX (enzyme:substrate ratio of 1∶100 (w:w)) showing that MMPs 1, 2, 8, 9 and 13 cleave LIX at position 4–5, whereas MMP-14 does not, and that only MMP-8 also cleaves at position 79∼80. Cleavage assays of rodent MMP-8 and MMP-13 are shown in the second 8 and 13 lanes. (B) Cleavage data are summarised using the full-length sequence of LIX.
Figure 4
Figure 4. In vitro cellular responses to MMP-8 cleaved LIX.
(A) Enhanced intracellular calcium mobilization was induced by LIX (5-92) and LIX (5-79) compared to full-length LIX (1-92) in recombinant CXCR2-expressing B300-19 cells and (B) PMNs isolated from Mmp8-/- mice (100 nM chemokine). (C) By transwell cell migration assay, both LIX (5-92) and LIX (5-79) truncated forms are more potent chemoattractants compared with the full-length LIX (1-92) for both CXCR2-expressing B300-19 cell transfectants and (D) murine PMNs isolated from either Mmp8+/+ or Mmp8-/- mice, all at 10 nM chemokine concentration.
Figure 5
Figure 5. MMP-8 is required for PMN chemotaxis towards LIX in vivo, but is not required for PMN cell migration.
PMN infiltration was greatly reduced in response to full-length LIX (1-92) injected into the dorsal skin air pouch of Mmp8-/- mice (black bars) compared to wild type mice (white bars). PMN numbers were calculated from myeloperoxidase assay after sacrifice at 0, 4, 8 and 12 h following injection of chemokine (n = 4). Unaltered PMN cell migration into air pouches of Mmp8-/- mice (black bars) compared to wild type mice (white bars) injected with MMP-cleaved analogues of LIX (5-92 or 5-79) reveals no intrinsic cell kinesis defects or chemotactic ability in the PMNs of the knock out mice.
Figure 6
Figure 6. MMP processing of CXCL8 and CXCL5.
(A) Tris-tricine 15% SDS-PAGE gel and MALDI-TOF mass spectrometry analysis of CXCL8 cleavage products after assay with the indicated human MMPs at an enzyme to substrate ratio of 1∶100. Cleavage assays of rodent MMP-8 are shown in the second 8 lane. The NH2-terminus of the CXCL8 truncated forms were deconvoluted from the mass spectrometry data and confirmed by Edman sequencing (as shown in C) with the CXCL8 forms identified shown below the corresponding gel lanes. 1-77, full-length CXCL8. (B) The effect of recombinant MMP-8 hemopexin C domain (MMP-8 CD), 10 µM EDTA, or 10 µM BB94 on CXCL8 cleavage by human MMP-8 as determined by Tris-tricine 15% SDS-PAGE and MALDI-TOF mass spectrometry. The m/z [M+H]+ of reaction products is as shown. (C) Identification of the NH2-termini of MMP cleavage products of CXCL8 and CXCL5 by MALDI-TOF mass spectrometry and NH2-terminal Edman sequencing. n.d., not determined. (D) Location of the various MMP cleavage sites on the CXCL8 and CXCL5 sequences.
Figure 7
Figure 7. Enhanced bioactivity of MMP-truncated CXCL8 in vitro and in vivo.
(A) Ca++ ion mobilization with 10 nM CXCL8 and (B) chemotaxis of CXCR2-transfected B300-19 cells stimulated with 10 nM full-length CXCL8 (1-77) and CXCL8 (6-77) synthetic analogue of MMP-8-cleaved CXCL8. (C) Time course of air pouch PMN influx in response to CXCL8 (1-77) and CXCL8 (6-77) in Mmp8-/- mice (black) and wild type (white) mice as quantitated by myeloperoxidase assay.
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