Limits...
Biochemical analysis of matrix metalloproteinase activation of chemokines CCL15 and CCL23 and increased glycosaminoglycan binding of CCL16.

Starr AE, Dufour A, Maier J, Overall CM - J. Biol. Chem. (2011)

Bottom Line: Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays.MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding.Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

ABSTRACT
Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.

Show MeSH
MMP-mediated cleavages of CCL15 and CCL23 activates chemokine agonism. Calcium mobilization (A–C) and chemotactic potential (D–E) of CCL15 and CCL23 are enhanced by MMP processing. A and B, representative traces of calcium flux, shown as a burst in relative fluorescence units (RFU) after the addition of 10 nm of full-length or MMP-truncated chemokine in CCR1-transfected B300-19 (A) and THP-1 cells loaded with Fluo-4 calcium indicator reagent (B). C, shown is the calcium mobilization dose response of THP-1 cells wherein calcium concentrations were calculated from relative fluorescence based on calibration with ionomycin and MnCl2. Transwell migration of CCR1-transfected B300-19 for 3 h (D) or THP-1 cells for 90 min (E) toward full-length or truncated chemokine through a 5-μm pore-sized filter is shown. Migrated cells were quantified by CyQUANT assay and displayed as chemotactic index, defined as the ratio of cells migrating in response to stimulus compared with buffer control. Results shown are the mean ± S.E. of three or more biological replicates of experiments completed in quadruplicate. Statistical analysis was by two-way ANOVA with Bonferroni post-test; the levels of significance comparing full-length to MMP-cleaved counterpart are: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3285354&req=5

Figure 6: MMP-mediated cleavages of CCL15 and CCL23 activates chemokine agonism. Calcium mobilization (A–C) and chemotactic potential (D–E) of CCL15 and CCL23 are enhanced by MMP processing. A and B, representative traces of calcium flux, shown as a burst in relative fluorescence units (RFU) after the addition of 10 nm of full-length or MMP-truncated chemokine in CCR1-transfected B300-19 (A) and THP-1 cells loaded with Fluo-4 calcium indicator reagent (B). C, shown is the calcium mobilization dose response of THP-1 cells wherein calcium concentrations were calculated from relative fluorescence based on calibration with ionomycin and MnCl2. Transwell migration of CCR1-transfected B300-19 for 3 h (D) or THP-1 cells for 90 min (E) toward full-length or truncated chemokine through a 5-μm pore-sized filter is shown. Migrated cells were quantified by CyQUANT assay and displayed as chemotactic index, defined as the ratio of cells migrating in response to stimulus compared with buffer control. Results shown are the mean ± S.E. of three or more biological replicates of experiments completed in quadruplicate. Statistical analysis was by two-way ANOVA with Bonferroni post-test; the levels of significance comparing full-length to MMP-cleaved counterpart are: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Mentions: MMP-cleaved chemokines induced a significant calcium flux after CCL15-(25–92, 28–92) and CCL23-(26–99) treatment of human CCR1-transfected B300-19 cells (Fig. 6A). With no activity observed by full-length CCL15 and CCL23, this shows that MMPs activate these two chemokines. To confirm this surprising result, similar experiments were performed using THP-1 cells (Fig. 6B); again, MMP-processed chemokines caused a concentration-dependent increase in THP-1 cell intracellular calcium mobilization, whereas a very minor response to CCL15-(1–92) and CCL23-(1–99) only occurred at the highest concentration evaluated (Fig. 6C). Notably, calcium mobilization did not occur after stimulation with the 19-mer peptide corresponding to CCL23-(5–23). Finally, MMP-processing of CCL16 did not alter calcium mobilization (not shown).


Biochemical analysis of matrix metalloproteinase activation of chemokines CCL15 and CCL23 and increased glycosaminoglycan binding of CCL16.

Starr AE, Dufour A, Maier J, Overall CM - J. Biol. Chem. (2011)

MMP-mediated cleavages of CCL15 and CCL23 activates chemokine agonism. Calcium mobilization (A–C) and chemotactic potential (D–E) of CCL15 and CCL23 are enhanced by MMP processing. A and B, representative traces of calcium flux, shown as a burst in relative fluorescence units (RFU) after the addition of 10 nm of full-length or MMP-truncated chemokine in CCR1-transfected B300-19 (A) and THP-1 cells loaded with Fluo-4 calcium indicator reagent (B). C, shown is the calcium mobilization dose response of THP-1 cells wherein calcium concentrations were calculated from relative fluorescence based on calibration with ionomycin and MnCl2. Transwell migration of CCR1-transfected B300-19 for 3 h (D) or THP-1 cells for 90 min (E) toward full-length or truncated chemokine through a 5-μm pore-sized filter is shown. Migrated cells were quantified by CyQUANT assay and displayed as chemotactic index, defined as the ratio of cells migrating in response to stimulus compared with buffer control. Results shown are the mean ± S.E. of three or more biological replicates of experiments completed in quadruplicate. Statistical analysis was by two-way ANOVA with Bonferroni post-test; the levels of significance comparing full-length to MMP-cleaved counterpart are: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285354&req=5

Figure 6: MMP-mediated cleavages of CCL15 and CCL23 activates chemokine agonism. Calcium mobilization (A–C) and chemotactic potential (D–E) of CCL15 and CCL23 are enhanced by MMP processing. A and B, representative traces of calcium flux, shown as a burst in relative fluorescence units (RFU) after the addition of 10 nm of full-length or MMP-truncated chemokine in CCR1-transfected B300-19 (A) and THP-1 cells loaded with Fluo-4 calcium indicator reagent (B). C, shown is the calcium mobilization dose response of THP-1 cells wherein calcium concentrations were calculated from relative fluorescence based on calibration with ionomycin and MnCl2. Transwell migration of CCR1-transfected B300-19 for 3 h (D) or THP-1 cells for 90 min (E) toward full-length or truncated chemokine through a 5-μm pore-sized filter is shown. Migrated cells were quantified by CyQUANT assay and displayed as chemotactic index, defined as the ratio of cells migrating in response to stimulus compared with buffer control. Results shown are the mean ± S.E. of three or more biological replicates of experiments completed in quadruplicate. Statistical analysis was by two-way ANOVA with Bonferroni post-test; the levels of significance comparing full-length to MMP-cleaved counterpart are: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Mentions: MMP-cleaved chemokines induced a significant calcium flux after CCL15-(25–92, 28–92) and CCL23-(26–99) treatment of human CCR1-transfected B300-19 cells (Fig. 6A). With no activity observed by full-length CCL15 and CCL23, this shows that MMPs activate these two chemokines. To confirm this surprising result, similar experiments were performed using THP-1 cells (Fig. 6B); again, MMP-processed chemokines caused a concentration-dependent increase in THP-1 cell intracellular calcium mobilization, whereas a very minor response to CCL15-(1–92) and CCL23-(1–99) only occurred at the highest concentration evaluated (Fig. 6C). Notably, calcium mobilization did not occur after stimulation with the 19-mer peptide corresponding to CCL23-(5–23). Finally, MMP-processing of CCL16 did not alter calcium mobilization (not shown).

Bottom Line: Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays.MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding.Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

ABSTRACT
Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.

Show MeSH