PAR1 activation induces monocyte/macrophage migration but not proliferation.

<p>(A–B) Migration assay of primary mouse BM-derived macrophages and a human monocyte cell line (THP-1) to SFLLRN demonstrating chemotaxis to the PAR1 agonist in both cell-types (MCP1 positive control). (C–D) SFLLRN has no pproliferative effect on primary mouse BM-derived macrophages (72 h) or a mouse macrophage cell line (RAW 264.7, 24 h) plated at the two different starting cell densities shown. (graphs show mean + SEM; p values as shown.)</p

Hepatic collagen I mRNA expression <i>in vivo</i>.

<p>A considerable reduction in COL1A1 gene expression was found in PAR1(−/−) mice subjected to carbon tetrachloride (CCl₄) liver injury compared to WT controls (A). A corresponding reduction in COL1A1 gene expression of lower magnitude was seen WT mice transplanted with PAR1(−/−) bone marrow (PAR1(−/−) BMT) compared to WT controls transplanted with WT bone marrow (WT BMT) (B). (graphs show mean + SEM, normalised to hypoxanthine guanine phosphoribosyl transferase housekeeping gene expression and against respective controls, p values as shown.)</p

Hepatic PAR1 is up-regulated during liver injury and is expressed on liver macrophages.

<p>(A–B) In wild-type mice, there is a marked up-regulation of hepatic PAR1 (brown, ×100) after carbon CCl₄ injury on cells along the hepatic scars (black arrows) and within the lobule itself (black arrowheads). (C–E) PAR1 (green) is commonly co-localised to macrophages (C, F4/80, red), but only occasionally to myofibroblasts (D, αSMA, red) and hepatic endothelium (E, endomucin, red). (Fluorescence images at ×200 magnification, nuclei in blue.)</p

Absence of PAR1 signalling on BM-derived cells can confer protection against liver fibrosis.

<p>C57/BL6 WT mice transplanted with BM from PAR1 knockout donors (PAR1(−/−)BMT, D–F) acquired significantly less hepatic collagen (images A&D, sirius red, ×100; graph G), fewer activated myofibroblasts (images B&E, αSMA, brown, ×100; graph H) and fewer hepatic macrophages (images C&F, F4/80, brown, ×200; graph I) after CCl₄ injury compared to control mice transplanted with WT BM from C57/BL6 donors (WTBMT, A–C). (graphs show mean + SEM, p values as shown.)</p

Primer nucleotide sequences used for real-time quantitative RT-PCR.

<p>Col 1A1, collagen Iα1; HPRT, hypoxanthine guanine phosphoribosyl transferase.</p

Absence of PAR1 signalling on BM-derived cells is associated with a significant reduction in macrophage recruitment to the injured liver.

<p>Female C57/BL6 mice received BMT from male PAR1 knockout donors (PAR1(−/−)BMT, B&D) or from male C57/BL6 WT controls (WTBMT, A&C) before CCl₄ liver injury. In both groups, BM-derived myofibroblasts (αSMA, red, A&B) and hepatic macrophages (F4/80, red, C&D) are seen (examples indicated by white arrowheads, ×400 magnification). (E) Splenic tissue showing complete haematopoietic reconstitution of recipient mice (female) with male cells, validating the efficacy of the BMT protocol. In all panels BM-derived (male) cells are identified by Y chromosome <i>in situ</i> hybridisation (green dot, Y chrm), localised within nuclei (DAPI, blue). (F) Graph showing the relative proportion of hepatic myofibroblasts (αSMA) and macrophages (F4/80) of BM origin. There is a significant reduction of BM-derived macrophage infiltration into the liver with loss of PAR1 signalling. (n = 8 per group, mean + SEM, p values as shown.)</p

Details of primary antibodies used for immunohistochemistry.

<p>αSMA, α-smooth muscle actin; PAR1, proteinase activated receptor 1; SCMW, sodium citrate buffer (2.94 g/L) microwave,</p>*<p>used for total hepatic macrophage counting with light microscopy.</p>**<p>used for fluorescence immunodetection of macrophages in conjunction with <i>in situ</i> hybridisation for Y chromosome.</p>§<p>a kind gift from Dr Eleanor Mackie, Melbourne, Australia.</p

Neutrophil chemotaxis to preterm BALF.

<p>Chemotaxis of adult human neutrophils to pooled BALF from BPD infants after incubation for 60 min. Conditions are detailed on the x-axis while concentration of neutrophils (cells/ml) are expressed on the y-axis. Bars are at means (± SEM) of three independent experiments, each done in triplicate. Conditions were compared by one-way ANOVA with Dunnet's correction, comparing against a control column (BALF only). (*** = p<0.001).</p

Modulation by proteinase-3.

<p>Summary of processing of rhIL-8₇₇ by purified proteinase-3 showing recovery of total IL-8 (squares) and IL-8₇₇ (circles) and release of MMP-9 from neutrophils in response to the products of conversion (triangles). Time (hours) is represented on the x-axis while IL-8/MMP-9 (fold change from 0-hour values) is represented on the y-axis. Points plotted are means (± SEM) of three independent experiments, each measured by ELISA in duplicate. Statistical differences in concentration compared to buffer-control at each time-point was tested by 2-way ANOVA with Bonferroni's correction. (* = p<0.05, ** = p<0.01, *** = p<0.001).</p

Patient and clinical characteristics.

<p>*Values shown are medians (inter-quartile range) with superscript numbers indicating days in addition to gestation in weeks; ^∧ values shown are numbers of infants (percentage of total in group).</p><p>Patient and clinical characteristics.</p