Cyclophilin C-associated protein: a normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo

Abstract

Mouse cyclophilin C-associated protein (CyCAP) is a member of the scavenger-receptor cysteine-rich domain superfamily and is 69% identical to the human Mac-2 binding protein. Here, we show that CyCAP is a widely expressed secreted glycoprotein that modulates the host response to endotoxin. Gene-targeted CyCAP-deficient mice are more sensitive to the lethal effects of endotoxin. In response to endotoxin, CyCAP-deficient mice overproduced interleukin 12 and interferon-gamma systemically and tumor necrosis factor alpha locally; these are proinflammatory molecules that also promote T helper 1 responses. Furthermore, macrophages stimulated in vitro with endotoxin in serum deficient in CyCAP secreted more tumor necrosis factor alpha, supporting the proposal that CyCAP specifically down-modulates endotoxin signaling.

Figure 1

Low-stringency Southern blot analyses. Southern blots of mouse genomic DNA digested with the indicated enzymes were hybridized with ³²P-labeled CyCAP and hMac-2-BP cDNA probes under low stringency as described in Materials and Methods.

Figure 2

Biosynthesis and secretion of CyCAP. (A) Pulse–chase analysis of CyCAP in MM55 kidney cells. Cells were pulsed for 10 min with [³⁵S]cysteine and [³⁵S]methionine and chased for the indicated times. CyCAP was immunoprecipitated from cell lysates and media with polyclonal rat serum against CyCAP. Control precipitations with normal rat serum were negative (data not shown). Proteins were resolved by using SDS/7.5% PAGE. Closed arrows represent positions of molecular-mass standards (Rainbow, Amersham Pharmacia). (B) CyCAP secretion from thioglycolate-elicited peritoneal macrophages. PECs were metabolically labeled overnight, and cypC-GST-glutathione agarose was used as an affinity reagent for CyCAP in the absence of CsA (lanes 3 and 5) or in the presence of CsA (lanes 4 and 6). Antibodies to macrophage markers Mac-1 and Mac-2 were used as positive controls (lanes 1 and 2).

Figure 3

Generation of CyCAP −/− mice. (A) The CyCAP genomic organization (Top), targeting vector (Middle), and targeted locus (Bottom). Exons are numbered 1–6, and restriction-enzyme sites are indicated. B, BamHI; H, HpaI. The expected size fragments for BamHI-digested DNA hybridized with the indicated 5′ probe for wild-type and targeted alleles are shown. (B) Southern blot analysis of representative tail DNA. Genomic tail DNA was digested with BamHI and hybridized with the 5′ probe indicated in A.

Figure 4

CyCAP −/− mice do not express CyCAP protein or RNA. (A) CyCAP is secreted from macrophages from wild-type (+/+) but not CyCAP-deficient (−/−) mice. Medium from metabolically labeled bone-marrow-derived macrophages was incubated with rat polyclonal serum to CyCAP (α-CyCAP) or normal rat serum (NRS). (B) CyCAP RNA is expressed in CyCAP +/+ but not CyCAP −/− mice. Northern blot analysis of total RNA from kidney and liver of wild-type and CyCAP-deficient mice probed sequentially with full-length ³²P-labeled CyCAP and cypA cDNA probes.

Figure 5

CyCAP-deficient mice are more sensitive to LPS. (A) CyCAP +/+ and CyCAP −/− (E14-159 line) mice were injected i.p. with LPS. (Squares) CyCAP +/+ mice (n = 8) injected with 10 μg/gbw LPS. (Diamonds) CyCAP +/+ mice (n = 9) injected with 7.5 μg/gbw LPS. (Circles) CyCAP −/− mice (n = 11) injected with 10 μg/gbw LPS. (Triangles) CyCAP −/− mice (n = 10) injected with 7.5 μg/gbw LPS. Survival was compared by using the Mantel–Cox log rank test. For the 10-μg/gbw dose, P = 0.0209; for the 7.5-μg/gbw dose, P = 0.0093. (B) LPS challenge of an independently derived CyCAP-deficient line (E14-274) with 10 μg/gbw LPS. (Squares) CyCAP +/+ mice (n = 7). (Circles) CyCAP −/− mice (n = 8).

Figure 6

In vivo cytokine levels in CyCAP −/− mice in response to LPS. Mice were challenged with 10 μg/gbw LPS, and cytokine levels were determined by ELISA. (A) Peak TNF-α levels in peritoneal lavages; three mice per group; P = 0.0056. (B) Peak IL-12 p70 serum levels 5 h after challenge. (C) Time course of IFN-γ production in serum. The dagger (†) indicates that the mouse died before the next time point. (D) Correlation between IL-12 p70 levels at 5 h and IFN-γ levels at 20 h; Y correlation coefficient, r = 0.942. (Diamonds) CyCAP-deficient mice. (Circles) Wild-type mice.

Figure 7

TNF-α release from PECs stimulated with LPS in the presence of serum from wild-type or CyCAP-deficient mice. (A) PECs from CyCAP-deficient mice were pooled and stimulated in vitro for 6 h with indicated concentrations of LPS in the presence of serum from CyCAP −/− (diamonds) or wild-type (squares) mice. TNF-α values represent means of duplicate samples ± SD. (B) PECs from eight CyCAP-deficient mice (cell numbers from 1 × 10⁶ to 5 × 10⁶ cells per ml) were stimulated with LPS (0.1 μg/ml) in serum from CyCAP +/+ or −/− mice, and TNF-α levels were measured. Data were analyzed by using a paired t test; P = 0.002.