Matrix Metalloproteinase-9-Generated COOH-, but Not NH2-Terminal Fragments of Serum Amyloid A1 Retain Potentiating Activity in Neutrophil Migration to CXCL8, With Loss of Direct Chemotactic and Cytokine-Inducing Capacity

Serum amyloid A1 (SAA1) is a prototypic acute phase protein, induced to extremely high levels by physical insults, including inflammation and infection. Human SAA and its NH2-terminal part have been studied extensively in the context of amyloidosis. By contrast, little is known about COOH-terminal fragments of SAA. Intact SAA1 chemoattracts leukocytes via the G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPR2). In addition to direct leukocyte activation, SAA1 induces chemokine production by signaling through toll-like receptor 2. We recently discovered that these induced chemokines synergize with intact SAA1 to chemoattract leukocytes in vitro and in vivo. Gelatinase B or matrix metalloproteinase-9 (MMP-9) is also induced by SAA1 during infection and inflammation and processes many substrates in the immune system. We demonstrate here that MMP-9 rapidly cleaves SAA1 at a known consensus sequence that is also present in gelatins. Processing of SAA1 by MMP-9 at an accessible loop between two alpha helices yielded predominantly three COOH-terminal fragments: SAA1(52–104), SAA1(57–104), and SAA1(58–104), with a relative molecular mass of 5,884.4, 5,327.3, and 5,256.3, respectively. To investigate the effect of proteolytic processing on the biological activity of SAA1, we chemically synthesized the COOH-terminal SAA fragments SAA1(52–104) and SAA1(58–104) and the complementary NH2-terminal peptide SAA1(1–51). In contrast to intact SAA1, the synthesized SAA1 peptides did not induce interleukin-8/CXCL8 in monocytes or fibroblasts. Moreover, these fragments possessed no direct chemotactic activity for neutrophils, as observed for intact SAA1. However, comparable to intact SAA1, SAA1(58–104) cooperated with CXCL8 in neutrophil activation and migration, whereas SAA1(1–51) lacked this potentiating activity. This cooperative interaction between the COOH-terminal SAA1 fragment and CXCL8 in neutrophil chemotaxis was mediated by FPR2. Hence, proteolytic cleavage of SAA1 by MMP-9 fine tunes the inflammatory capacity of this acute phase protein in that only the synergistic interactions with chemokines remain to prolong the duration of inflammation.

Serum amyloid A1 (SAA1) is a prototypic acute phase protein, induced to extremely high levels by physical insults, including inflammation and infection. Human SAA and its NH2terminal part have been studied extensively in the context of amyloidosis. By contrast, little is known about COOH-terminal fragments of SAA. Intact SAA1 chemoattracts leukocytes via the G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPR2). In addition to direct leukocyte activation, SAA1 induces chemokine production by signaling through toll-like receptor 2. We recently discovered that these induced chemokines synergize with intact SAA1 to chemoattract leukocytes in vitro and in vivo. Gelatinase B or matrix metalloproteinase-9 (MMP-9) is also induced by SAA1 during infection and inflammation and processes many substrates in the immune system. We demonstrate here that MMP-9 rapidly cleaves SAA1 at a known consensus sequence that is also present in gelatins. Processing of SAA1 by MMP-9 at an accessible loop between two alpha helices yielded predominantly three COOH-terminal fragments: SAA1(52-104), SAA1 , and SAA1(58-104), with a relative molecular mass of 5,884.4, 5,327.3, and 5,256.3, respectively. To investigate the effect of proteolytic processing on the biological activity of SAA1, we chemically synthesized the COOH-terminal SAA fragments SAA1  and SAA1  and the complementary NH2-terminal peptide SAA1 . In contrast to intact SAA1, the synthesized SAA1 peptides did not induce interleukin-8/CXCL8 in monocytes or fibroblasts. Moreover, these fragments possessed no direct chemotactic activity for neutrophils, as observed for intact SAA1. However, comparable to intact SAA1, SAA1(58-104) cooperated with CXCL8 in inTrODUcTiOn Serum amyloid A (SAA) is an acute phase protein, mainly produced in the liver under inflammatory conditions (1), but extrahepatic production of SAA has also been reported (2,3). Moreover, SAA is involved in many inflammatory diseases, such as rheumatoid arthritis, diabetes type 2 and cancer (4)(5)(6)(7). Several biological activities have been ascribed to SAA. At high concentrations (1-20 µg/ml), SAA has antiviral (8,9) and antibacterial (10)(11)(12) activities and plays a role in cholesterol transport (13). More relevantly, at low concentrations (10-500 ng/ml), SAA induces cytokines, chemokines, and matrix metalloproteinases (MMPs) (14)(15)(16)(17). One of the most important biological activities of SAA, exerted at similarly low concentrations, is its direct and indirect chemotactic activity for monocytes, neutrophils, immature dendritic cells, and regulatory T cells (17)(18)(19)(20)(21). On one hand, the direct chemotactic activity of SAA is mediated by the G proteincoupled receptor (GPCR) formyl peptide receptor 2 (FPR2) (22). On the other hand, SAA is indirectly chemotactic for leukocytes via induction of chemokines through binding to toll-like receptor 2 (TLR2) (17,21). However, other receptors are also involved in cytokine induction by SAA variants (23)(24)(25). Following induction by serum amyloid A1 (SAA1), these chemokines synergize with each other, but also with SAA, to enhance leukocyte migration to the inflammatory site (17). Chemokines are small chemotactic cytokines, directing leukocyte migration under homeostatic and inflammatory conditions through binding to GPCRs (26). The chemokine family mainly consists of CC and CXC chemokines, depending on the position of the first two cysteine residues in their primary structure (27). Posttranslational truncation of chemokines can increase, decrease, or even completely block their chemotactic capacity (27,28).
Several variants of SAA exist: SAA1, SAA2, SAA3, and SAA4. SAA1 and SAA2 are highly induced during the acute phase response (100-to 1,000-fold increase), whereas SAA4 is constitutively present in plasma at lower concentrations (3). Furthermore, truncated forms of circulating SAA have been detected in several diseases (29,30). Proteolytic cleavage of SAA occurs through interaction with mostly MMPs (i.e., MMP-1, -2, and -3) (31, 32), but SAA can also be cleaved by other proteases (33). Nonetheless, the role of the different SAA variants and their processed forms in the pathogenesis of diseases and their potential distinct functions still need to be elucidated. We recently demonstrated that posttranslational cleavage of SAA variants by proteases may lead to alterations in their biological activities. Indeed, SAA1(47-104) cooperates with chemokines in neutrophil and monocyte migration and desensitizes the synergy between intact SAA1 and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds FPR2 (34). As its name suggests, SAA has been studied in inflammation-associated amyloidosis. However, being an acute phase reactant, SAA1 is present in the circulation and in exudates during infection or inflammation, even when no amyloidosis occurs. Matrix metalloproteinase-9 (MMP-9) or gelatinase B is a tuner and amplifier of immune functions (35), and its levels are increased in all inflammatory diseases (36).

MaTerials anD MeThODs reagents
Full length human proMMP-9/gelatinase B was expressed in Sf9 insect cells and purified to homogeneity by gelatin-Sepharose chromatography. ProMMP-9 was activated with the catalytic domain of MMP-3 (Merck Millipore, Darmstadt, Germany) as described (37). Afterward, MMP-9 activity was confirmed by SDS-PAGE of cleaved substrate and a gelatin degradation assay as previously described (38). Human (hu) recombinant intact apo-SAA1 (rSAA1α) containing an NH2-terminal methionine (11,814 kDa), CXCL8(6-77), and recombinant IL-1β were purchased from Peprotech (Rocky Hill, NJ, USA). Lipopolysaccharide (LPS) from Escherichia coli (0111:B4) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The selective FPR2 antagonist WRW4 and the FPR2 agonist WKYMVm were purchased from Calbiochem (San Diego, CA, USA) and Phoenix Pharmaceuticals (Burlingame, CA, USA), respectively. neutrophil activation and migration, whereas SAA1(1-51) lacked this potentiating activity. This cooperative interaction between the COOH-terminal SAA1 fragment and CXCL8 in neutrophil chemotaxis was mediated by FPR2. Hence, proteolytic cleavage of SAA1 by MMP-9 fine tunes the inflammatory capacity of this acute phase protein in that only the synergistic interactions with chemokines remain to prolong the duration of inflammation.

neutrophilic granulocyte activation assays
Neutrophil migration was measured in the Boyden microchamber assay (Neuro Probe, Gaithersburg, MD, USA) as previously described (21). The chemotactic index (CI) was calculated by dividing the average number of cells migrated to the chemotactic factor by the average number of cells migrated to the chemotaxis control buffer. Synergy was obtained when the net chemotactic index (net CI = CI − 1) of the combined chemotactic substances was significantly higher than the sum of the net CI of the chemotactic substances added separately to the microchamber.
For antagonizing experiments, the upper wells of the Boyden microchamber were loaded with neutrophils in the presence of the FPR2 antagonist WRW4 (20 µg/ml). The shape change test was used to measure fast and direct activation of neutrophils in suspension. Purified human neutrophils (0.6 × 10 6 cells/ml) were diluted in Hanks' balanced salt solution (HBSS; Life Technologies, Paisley, UK) supplemented with 10 mM HEPES (Life Technologies) and incubated in a 96-well microtiter plate in the presence of dilution buffer, CXCL8, SAA1(58-104), or a combination of CXCL8 and SAA1 . After 3 min of stimulation, the cells were fixed by adding an equal volume of HBSS/HEPES buffer containing 4% formaldehyde. For each condition, 100 cells, morphologically identified as round, blebbed, or elongated cells, were counted microscopically (magnification 200×) and independently by two individuals in a blinded manner. For the assessment of cooperation, the net percentages of neutrophils undergoing shape change (blebbed + elongated cells) were used.

chemokine induction experiments
Monolayers of primary human fibroblasts were grown to confluency in 48-well plates in MEM containing 10% FCS. Cells were stimulated in MEM containing 2% FCS for 24 h with different doses of IL-1β, SAA1α, the COOH-terminal, and NH2-terminal SAA1 peptides or were left untreated (control). CD14 + monocytes were seeded in 48-well plates in RPMI 1640 medium supplemented with 0.5% human serum albumin (Belgian Red Cross, Brussels, Belgium) at a concentration of 2 × 10 6 cells/ ml and induced for 24 h with different doses of LPS, SAA1α, SAA1(1-51), SAA1(52-104), and SAA1(58-104) or were left untreated (control). Levels of human CXCL8 were quantified by a specific sandwich ELISA developed in our laboratory (lowest level of detection: 0.04 ng/ml) (41).

statistical analysis
Data were first analyzed by the non-parametric Kruskal-Wallis test (Statistica 12.0) for comparison of multiple groups before performing pairwise comparisons. The Mann-Whitney U test and the Wilcoxon Sum Rank test were used to compare data from two unpaired or paired data sets, respectively. resUlTs cleavage of saa1 by gelatinase B/MMP-9 To determine whether SAA1 is cleaved by gelatinase B/MMP-9, recombinant intact SAA1 (11.8 kDa) was incubated at 37°C with the enzyme at a 1:50 enzyme:substrate ratio and analyzed by SDS-PAGE. Figure 1 shows a clear shift of the protein mass toward 5 kDa reaction products after incubation of SAA1 with MMP-9, demonstrating that MMP-9 processes the acute phase protein SAA1, presumably by cleaving it in the middle of the protein. The kinetics of the cleavage of SAA1 by MMP-9 were studied by taking samples at various time intervals during the incubation (Figure 1). Cleavage of SAA1 by MMP-9 resulted in a decrease of the intensity of intact SAA1 over time. The cleavage products were already visible after 0.25 h, and the intensity of the SAA1 cleavage products increased over time. However, MMP-9 treatment did not lead to complete degradation of intact SAA1 after 30 h.
To determine the exact cleavage sites of MMP-9 in SAA1, intact SAA1 was incubated with the enzyme and subsequently analyzed by MS (Figure 2). After 3 h, intact SAA1 (Figures 2A,B) was cleaved by MMP-9 into three different COOH-terminal fragments: SAA1(52-104), SAA1(57-104), and SAA1(58-104)  ( Figures 2C,D). The relative intensities of the different peptide peaks are indicated in Table 1. The cleavage thus occurred after a glycine residue at position 51 and yielded a COOH-terminal part of similar size starting with valine at position 52. This cleavage site is within a small protein loop between two alpha helices of SAA1 (42) and coincides with the sequon Gly-Pro-Xaa-Gly-hydrophobic residue, which is the preferred consensus sequence and cleavage site of MMP-9 in denatured collagens or gelatins (43).

DiscUssiOn
Proteolytic processing of inflammatory mediators is a common feature to control the innate immune response. Indeed, The cooperative effect Between cXcl8 and the cOOh-Terminal Peptides of saa1 in neutrophil chemotaxis is inhibited by the selective FPr2 antagonist WrW 4 To investigate whether the cooperation between the COOHterminal SAA1 peptides and CXCL8 in neutrophil chemotaxis implies the binding of SAA1(58-104) to the SAA1 receptor FPR2, the combination of SAA1(58-104) and CXCL8 was evaluated in the microchamber assay in the presence of the specific FPR2 antagonist WRW4 (Figure 8). In the absence of the FPR2 antagonist, SAA1(58-104) at 3,000 ng/ml (CI = 1.9 ± 0.7) cooperated (CI = 49.8 ± 3.6; n = 4; p = 0.03) with CXCL8 at 3 ng/ ml (CI = 31.0 ± 4.6). Treatment of neutrophils with WRW4 at inflammation is steered by proteases cleaving cytokines, chemokines, acute phase proteins, etc. Some cytokines such as IL-1 are naturally produced as precursor proteins which need to be enzymatically processed. The generated fully active endogenous pyrogen induces acute phase proteins, chemokines, and other cytokines in various cell types including leukocytes, fibroblasts, hepatocytes, and endothelial cells (45)(46)(47). Chemokines, which are not produced as pro-peptides, are confronted with many different immune cell-derived enzymes resulting in a complex chemokine-protease interacting network (28,48). For example, soluble or membrane-associated CD26/DPP4 can NH2-terminally truncate most inflammatory chemokines with a different outcome on their biological effects. Indeed, the CXCR3 ligands lose their chemotactic activity upon removal of their two NH2-terminal residues, whereas such truncation renders some CC chemokines (e.g., CCL3) more active on specific leukocyte types (27,28). Furthermore, chemokines such as CXCL8 stimulate neutrophils to secrete proteases such as gelatinase B/MMP-9, which in turn cleaves CXCL8 into a more active neutrophil chemoattractant (49). As a consequence, proteolytic processing of chemokines can enhance or dampen the inflammatory response (28). Similarly, the acute phase protein SAA1 can induce MMP-9 (50) which can enzymatically cleave SAA1 (Figures 1 and 2). However, the biological consequences of SAA1 processing are poorly studied (31,32,51). Several functions have been ascribed to SAA, of which chemotaxis of leukocytes, induction of chemokines and matrix degrading enzymes reach the highest specific activity (3,17,29,50,(52)(53)(54)(55). Moreover, both MMPs (e.g. MMP-9) and chemokines (e.g. CXCL8 and CCL3) are co-induced in monocytes by SAA1 (17,50). Furthermore, truncated CXCL8 can synergize with SAA1 in leukocyte chemotaxis (17,21,49).
Little is known about the role of MMP-9-generated COOHterminal fragments of SAA1 in pathology, whereas the literature on NH2-terminal fragments is more comprehensive (29,30,32,(58)(59)(60). In particular MMPs are detected during amyloidosis in amyloid A (AA) deposits together with NH2-terminal fragments of SAA1 (51). Amyloidosis secondary to chronic inflammatory diseases, such as rheumatoid arthritis, is caused by the systemic deposition of insoluble AA fibrils in various organs (29,32). SAA is considered as the precursor of AA fibril protein deposited during this disease (29,50,61,62). The amyloid fibrils found in patients with AA amyloidosis largely consist of SAA(1-76), as the predominant AA protein, although NH2-terminal fragments of different lengths have been reported (31,51,58,(63)(64)(65). However, the NH2terminal SAA1(1-51) fragment, investigated in this paper, has not been described in the literature as part of AA amyloid deposits.
In conclusion, we demonstrated that the inflammationassociated MMP-9 generates SAA1 COOH-terminal fragments with impaired TLR2-mediated chemokine-inducing capacity, while retaining their FPR2-mediated potential to cooperate with chemokines in leukocyte activation and attraction. The TLR2mediated cytokine-inducing capacity of SAA1 assists in initiating an inflammatory response, but later on in this process, when MMPs are released, the inflammatory capacity of SAA1 is finetuned and only the cooperative interactions with chemokines remain to prolong the duration of inflammation.

eThics sTaTeMenT
This study was carried out in accordance with the study protocol (S58418) that was approved by the ethical committee of the KU Leuven with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. aUThOr cOnTriBUTiOns MG: planned and performed experiments, analyzed data, wrote part of the manuscript and submitted the manuscript; MDB: planned and performed experiments, analyzed data, wrote part of the manuscript; SAS: performed experiments, analyzed data and corrected the manuscript; JV: was involved in proteolytic cleavage of SAA; SK, NP, LV and NB: performed experiments; GO: was involved in proteolytic cleavage of SAA and corrected the manuscript; PP: executed biochemical quality control of reagents, performed protein synthesis and corrected the manuscript; JVD: analyzed data, designed the study, corrected the manuscript; SS: designed the study, gave technical advice, analyzed data, corrected the manuscript.

acKnOWleDgMenTs
The authors would like to thank Rik Janssens, Isabelle Ronsse, Anke Renders, and Melissa Stas for their technical assistance.