Latency-associated peptide (LAP) induces monocyte chemotaxis via a pathway distinct from TGF-β1.

<p>C57BL/6 female mice were anesthetized and subcutaneously injected with Matrigel™ matrix supplemented with PBS, rhLAP or CCL2/MCP-1. After 10 days the mice were sacrificed, skinned, and the plugs were removed, fixed and embedded in paraffin then stained for CD68+ cells (mononuclear phagocytes). Matrigel matrix supplemented with (A) PBS, (B) 10 pg/ml rhLAP, or (C) 10 ng/ml CCL2 were assessed for CD68+ cell recruitment and then quantified by blinded counts (D). Arrow heads indicate CD68+ cells. (*p<0.05 for condition vs. PBS control plugs, n = 3 for CCL2 and n = 2 for LAP) Human monocytes (5×10⁴/condition) were suspended in Gey's balanced salt solution and assayed for their chemotactic response to rhLAP. Recruitment was assayed by incubating monocytes in modified Boyden chambers for 90 minutes at 37°C and quantified by counting five high-powered fields of stained membranes in a blinded fashion. (E) Increasing doses of rhLAP (0-100 pg/ml) were used to stimulate monocyte recruitment (†p<0.001 from the non-stimulated sample, n = 7). (F) Analysis for evidence of directed recruitment of monocytes by LAP was assessed by exposing monocytes to a “true” gradient of rhLAP (10 pg/ml) (rhLAP in the chamber opposite the monocytes <i>only</i>; dark bars–“Monocytes”) or no gradient (rhLAP on both sides of the chamber; lighter bars–“Monocytes+LAP”). These data represent two independent studies done in triplicate (*p<0.001 vs. mono + LAP condition). (G) Assessment of LAP-specific chemotaxis was performed by pre-incubating cell suspensions with the SB 431542 hydrate from Sigma (St.Louis, MO), a selective inhibitor of TGF-β type 1 receptor kinases (Activin Receptor-Like Kinases, ALK-4,-5, and-7) 20 minutes prior to loading on chemotaxis chambers. Monocytes were then exposed to optimal chemotactic doses of LAP (10 pg/ml) and TGF-β1 (100 pg/ml). All bars represent the mean±SEM for n = 4 samples (* p<0.05 by ANOVA with post-hoc testing).</p

LAP-induced monocyte chemotaxis is blocked by specific inhibitors of LAP-TSP-1 interactions.

<p>Human monocytes (5×10⁴/condition) were suspended in Gey's balanced salt solution and assayed for their chemotactic response to rhLAP. Recruitment was assayed by incubating monocytes in modified Boyden chambers for 90 minutes at 37°C and quantified by counting five high-powered fields of stained membranes in a blinded fashion and reported as fold-increase of LAP (10 pg/ml)-induced monocyte chemotaxis compared to monocyte chemotaxis induced by media alone. rhLAP at 10 pg/ml was chosen based on previous experiments that determined this dose induced maximal cellular recruitment. These data represent n = 3 (minimum) separate experiments expressed as the mean fold-change±SEM. Reported p values compare fold-change in monocyte chemotaxis induced by LAP without and with identified inhibitors.</p

LAP-induced inflammatory cell recruitment is altered in TSP-1 deficient (TSP-1 −/−) mice.

<p>(A) Bone marrow macrophages from wild type C57BL/6 or TSP-1−/− mice were isolated and assessed for chemotaxis activity using modified Boyden chambers. Macrophages (5×10⁴/condition) were suspended serum-free in Gey's balanced salt solution and chemotaxis was assayed by counting the mean of five blinded high-powered fields on the polycarbonate filter (†p<0.001 for the chemotaxis of wild-type vs. TSP-1 −/− BMM). The data is expressed as fold-change in the mean compared to control (media alone)±SEM for two independent studies done in triplicate. (B) LAP's ability to affect the direct DTHR was tested in TSP-1 −/− and wild-type mice. TSP-1 −/− and wild-type C57BL/6 mice were sensitized to tetanus toxin (TT) and then tested to confirm they had developed a TT-induced DTHR. This direct DTHR assay was performed in the pinnae of these mice with simultaneous injection (35 µl) of antigen (tetanus toxin) alone or with rhLAP (10 pg) (†p = 0.004 for LAP-treated versus untreated WT mice, n = 3). All bars represent the mean±SEM for two studies unless otherwise noted. DTHR, delayed-type hypersensitivity response; TT, tetanus toxin; WT, wild type, BKG, background ear girth.</p

LAP is able to suppress <i>in vivo</i> cellular inflammation similar to TGF-β1 through interactions with thrombospondin-1.

<p>A transfer DTHR assay was performed with antigen (tetanus toxin) and tested agents (TGF-β1, rhLAP and anti-TGF-β1, LAP or TSP-1). C57BL/6 mice were sensitized to tetanus toxin (TT) and then tested to confirm they had developed a TT-induced DTHR. DTHR was induced in the pinnae of naïve C57BL/6 mice with simultaneous injection (35 µl) of antigen and splenocytes (8×10⁶ cells/condition) harvested from antigen-sensitized mice. DTHR was quantified by measurement of ear thickness 24 hours after injection. (A) LAP (10 pg) and TGF-β1 (5 ng) were tested for the ability to reduce DTHR when used alone, with anti-TGF-β1 antibodies (1 µg) or anti-LAP antibodies (1 µg) (*p<0.02 versus tetanus sensitized splenocytes + TT; †p<0.01 versus tetanus sensitized splenocytes + TT, n = 5). Similar results were observed when this assay was repeated using C57BL/6 mice that had rejected a cardiac allograft (DBA/2->C57BL/6) as a model of transfer DTHR. (B) Anti-TSP-1 antibody or isogenic control IgG (all at 1 µg) were tested for their ability to interfere with rhLAP suppression of TT-induced DTHR (*p<0.02 versus tetanus sensitized splenocytes + TT; †p<0.01 versus tetanus sensitized splenocytes + TT, n = 3). (C) Transfer DTHR assay was performed with antigen, rhLAP and the inhibitory peptide, LSKL or its scrambled control SLLK (20 µM) (†p<0.01 versus tetanus-sensitized splenocytes + TT, n = 2). (D) To determine if LAP used an IL-10 dependent pathway to inhibit the DTHR (as previously published) the DTHR transfer assay was repeated with anti-IL-10 antibodies and isotype control. (1 µg each) (*p<0.01 vs. tetanus-sensitized splenocytes + TT, n = 3) All bars represent the mean±SEM. DTHR, delayed-type hypersensitivity response; Ag, Antigen; BKG, background ear girth.</p