Extracellular cyclophilins contribute to the regulation of inflammatory responses

Abstract

The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.

FIGURE 1

CD147 expression on murine leukocytes. A, Leukocytes obtained from peripheral blood of C57BL/6 mice were stained with anti-CD147 mAb, or isotype control mAb, followed by FITC-conjugated anti-rat IgG. Analysis gates were set on the lymphoid, monocytic, or granulocytic population using forward scatter/side scatter distributions.■, Isotype control staining;□, CD147 expression. B, Neutrophils from different sites were compared for CD147 expression. Each population was stained with anti-CD147 or isotype control mAb, followed by FITC-anti-rat IgG. Neutrophils were identified by additional staining with PE-conjugated anti-GR-1 mAb. Data show the expression of CD147 (□) relative to isotype control staining (■) after gating on GR-1⁺ leukocytes.

FIGURE 2

Murine neutrophils migrate in response to CyPA in vitro. Neutrophils were enriched from the peritoneal cavity of C57BL/6 mice and set up in 48-well Boyden chambers in the presence of recombinant CyPA, FMLP (as a positive control), or medium alone. A, Different concentrations of CyPA were compared, relative to 10⁻⁷ M FMLP. B, A dose of 100 ng/ml CyPA was used with or without the addition of 25 µg/ml anti-CD147 or isotype control mAb. C, A dose of 100 ng/ml CyPA or 10⁻⁷ M FMLP was used with or without the addition of 25 µg/ml anti-CD147 or 2 µM nonimmunosuppressive CsA (SDZNIM811). Graphs show the mean (±SE) chemotactic index (number of cells migrating in response to chemotactic agent divided by number of cells migrating to medium alone) for each group (n = 4–6 wells/group). Student’s t test was used to establish statistical significance between groups of wells treated with anti-CD147 or CsA compared with CyPA alone: ***, p < 0.001.

FIGURE 3

Inflammation induced by intranasal LPS is associated with elevated cyclophilins in airways. Groups (n = 5) of C57BL/6 mice were given PBS or 2 µg of LPS by intranasal delivery; 18 h later, the animals were killed, and their BAL fluid was collected. A, For Western blot analysis, BAL fluid was cleared of leukocytes by centrifugation and pooled within groups of mice. Equivalent volumes of each pool were run under denaturing conditions by SDS-PAGE, followed by transfer onto nylon membranes and then probing with rabbit-anti-CyPA. The Western blot shows detection of CyPA in groups of mice from two or three individual experiments. The standard was a lysate of HEK293T cells. B, Densitometry was conducted on the individual lanes from the blot. Data show the mean gel band density (GBD) for the lanes from PBS vs LPS groups. Student’s t test was used to establish statistical significance:*, p < 0.05. C, Neutrophil numbers were determined in each of the pooled BAL samples by staining cell suspensions with PE-conjugated anti-GR-1, followed by FACS analysis. Data show a correlation between the number of neutrophils recovered and the CyPA content (based on GBD) for each BAL sample. A correlation coefficient (r²) was calculated between the two parameters.

FIGURE 4

Inhibition of cyclophilin-CD147 interactions by in vivo anti-CD147 treatment significantly reduces tissue inflammation. A, Groups of C57BL/6 mice were given an i.p. injection of 100 µg of anti-CD147 or isotype control mAb 1 day before treatment with LPS and then a second injection at the same time as intranasal delivery of LPS. At 18 h after LPS delivery, the mice were killed, and their lung tissue and BAL fluid were collected. Tissue and BAL leukocytes from individual mice were stained with PE-anti-GR-1 and analyzed by FACS. Data show the mean number (±SE) of neutrophils detected in each group of mice (n = 7–9/group). Student’s t test was used to establish statistical significance:***, p < 0.001; *, p < 0.05. B, In some studies lung tissue was fixed in 10% formalin, sectioned, and stained with H&E for histological analysis. Histology (×10 magnification) shows representative sections from the following groups: a, PBS alone; b, LPS alone; c, LPS plus isotype treatment; d, LPS plus anti-CD147 treatment. Arrows denote foci of inflammation.

FIGURE 5

Inhibition of cyclophilins by in vivo CsA treatment significantly reduces tissue inflammation. A, Groups of C75BL/6 mice were given an i.p. injection of 20 mg/kg body weight SDZNIM811 (or diluent alone), and 10 h later a second dose was given i.p. at the same time as intranasal delivery of LPS. All groups were killed 18 h later, and lung tissue was isolated and stained with PE-anti-GR-1 for FACS analysis. Data show the mean number (±SE) of neutrophils detected in lung tissues of each group of mice (n = 5/group). B, Groups of mice were given either two doses of 20 mg/kg SDZNIM811 (10 h before intranasal LPS and concurrently with LPS), two doses of 100 µg of anti-CD147 mAb (1 day before intranasal LPS and concurrently with LPS), or both treatments. All groups were killed 18 h later, and lung tissue was isolated and stained with PE-anti-GR-1 for FACS analysis. Data show the mean number (±SE) of neutrophils detected in each group of mice (n = 8–10/group). For all studies, Student’s t test was used to establish statistical significance: ***, p < 0.001;**, p < 0.01; *, p < 0.05.