Human S100A9 induced THP-1 cell migration requires MEK/ERK and PI3K but not p38.

<p>Chemotactic response of THP-1 towards hS100A9 (1 ng/ml) in the absence or presence of specific inhibitors targeting the MEK/ERK pathway (A), the PI3 kinase pathway (B), the NF-κB pathway (C), and the p38 MAPK pathway (D). The inhibitor concentrations (μM) are indicated. Data shown is mean±SD of triplicate samples from a representative of three independent experiments.</p

Murine S100A9 induced lung inflammation is RAGE-independent.

<p>Wild type C57Bl/6 or C57Bl/6 <i>ager</i>-/- mice were challenged intranasally with PBS, or adeno-null or adeno-murine S100A9, after 8 days, mice were sacrificed and BAL fluids or lung tissue were collected for analysis. (A) Cells were obtained from the BAL fluids using cytospins, stained with diff-quik and the total cell counts, neutrophil counts and macrophage counts were recorded. (B) mIFNγ and mIL-6 proteins levels found in BAL fluids of wild type and <i>ager</i>-/- mice. (C) Western blot analysis of murine S100A9 levels in BAL fluids. (D) H&E staining of paraffin fixed lung tissue (left panel), and pathology scores (right panel). Non-parametric Mann-Whitney test was used to determine statistical difference between two groups.</p

S100A9 Induced Inflammatory Responses Are Mediated by Distinct Damage Associated Molecular Patterns (DAMP) Receptors <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses <i>in vitro</i>. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation <i>in vivo</i>. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response <i>in vivo</i>, and despite the capacity of RAGE and TLR4 blockade to inhibit responses <i>in vitro</i>, the response is predominately independent of both these receptors.</p></div

Murine S100A9 induced inflammatory infiltrates are TLR4-independent.

<p>Adeno-murine S100A9, Adeno-null or PBS was administered intranasally into wild-type (C3H/HeOuj) and TLR4-defective (C3H/HeJ) mice. After 10 days, mice were euthanized and BAL fluids or lung tissue was collected for analysis. (A) Cells were obtained from the BAL fluids using cytospins, stained with diff-quik and the total cell counts, neutrophil counts and macrophage counts were recorded. (B) mIFNγ and mIL-6 expression in BAL fluid in wild type and TLR4 defective mice. (C) Western blot analyses of mS100A9 expression in BAL fluid in wild type and TLR4 defective mice. (D) H&E staining of paraffin fixed lung tissue (left panel), and pathology scores (right panel). Non-parametric Mann-Whitney test was used to determine statistical difference between two groups.</p

RAGE blockade inhibits calgranulin (hS100A8, hS100A9 or hS100A12) induced THP-1 cell migration but does not impact migration of all S100s.

<p>Migration indices for THP-1 cells in response to hS100A8 (A), hS100A9 (B), and hS100A12 (C). Effects of anti-RAGE, anti-hTLR4 Abs and isotype control Abs on hS100A8 (D), hS100A9 (E) and hS100A12 (F) induced THP-1 cell migration. Serial dilution of anti-RAGE, anti-TLR4 or isotype-matched control Ab were incubated with THP-1 cells in the upper wells of the chemotaxis chamber, and optimal amounts of hS100A8 (1 ng/ml), hS100A9 (1 ng/ml) and hS100A12 (100 ng/ml) were added in the lower wells. Percentage inhibition is relative to no Ab treatment. One representative of three independent experiments is shown (mean ±SD of triplicate wells) (G) The effects of a fixed 10 μg/ml dose of anti-RAGE, anti-hTLR4 and control Abs on THP-1 cell migration mediated by other S100s and MCP1. The S100s were used at their optimal concentrations (indicated on graphs). The maximal chemotactic indexes were S100A1 (2.0), S100A4 (2.9), S100A6 (2.9), S100A7 (3.7), S100A8/A9 (3.1), S100A10 (1.9), S100A14 (2.7), S100A16 (2.4), S100P (3.5), S100B (3.5) and MCP1 (9.5). Percentage inhibition is relative to no Ab treatment. Mean inhibition ± SD for one of two independent experiments is shown. Unpaired T-test was used to determine if the anti-RAGE or anti-hTLR4 Ab treatments were significantly different from the isotype control Ab (*P<0.05, **P<0.01, ***P<0.001).</p

TLR4 but not RAGE is necessary for calgranulin-mediated proinflammatory cytokine induction, but other S100s exhibited variable cytokine responses.

<p>Human PBMCs were stimulated with 10 μg/ml of human S100 preparations and levels of IL-6 (and other cytokines) were measured after 16 h in culture. (A) Cells were stimulated with low endotoxin preparations of calgranulins (S100A8, S100A9 or S100A12) or LPS and were treated with either nil, control Ab, anti-hTLR4, anti-RAGE Abs or Polymyxin B (PMB). (B) Cells were stimulated with S100A1, S100A16, S100B and S100A8/A9 and were similarly treated with nil, control Ab, anti-hTLR4 Ab, anti-RAGE Ab, or Polymyxin B. Representative data from one of three experiments is shown. Individual experiments were performed in triplicate and mean ± SD are given. Unpaired T-tests was used to determine if the test antibody differed significantly from their respective control Abs following stimulation, and if the polymyxin treatment differed from no treatment following stimulation (*P<0.05, **P<0.01, ***P<0.001).</p

Human S100A9 induced migration of leukocyte populations is inhibited by anti-RAGE Ab but not anti-TLR4 Ab.

<p>Chemotactic responses to S100A9 of human leukocyte populations were examined at the optimal concentration (1 ng/ml). The effects of TLR4 and RAGE blockade on S100A9-mediated migration of granulocytes (A), monocytes (B), and lymphocytes (C), with 10 μg/ml of anti-RAGE, anti-hTLR4 Ab and isotype control Abs. Data shown is mean±SD of triplicate samples from a representative of three independent experiments. Unpaired T-test was used to determine if the anti-RAGE or anti-TLR4 treatments were significantly different from the isotype control Ab (*P<0.05, **P<0.01, ***P<0.001).</p