Caspases and nitric oxide broadly regulate dendritic cell maturation and surface expression of class II MHC proteins - PubMed (original) (raw)

Caspases and nitric oxide broadly regulate dendritic cell maturation and surface expression of class II MHC proteins

Siew Heng Wong et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The passage of dendritic cells (DC) from immature to terminally differentiated antigen-presenting cells is accompanied by numerous morphological, phenotypic, and functional changes. These changes include, for example, expression of "empty" class II MHC proteins (MHCII) at the surface in immature DC, whereas a much larger amount of peptide-loaded MHCII is expressed at the surface in mature DC. Here we show that, in cultured immature DC derived from murine bone-marrow precursors, a number of molecules involved in intracellular trafficking were present in a cleaved form, degraded by caspase-like proteases. Cleavage was either inhibited or reduced significantly during maturation of DC induced by either LPS and TNF-alpha or by peptides that inhibit caspase activities. Inducible nitric oxide (NO) synthetase up-regulated by LPS was essential for inhibiting the caspase-like activity during the maturation of DC. Moreover, treatment with LPS or caspase inhibitor resulted in expression of MHCII/peptide complexes at the cell surface. Thus, the alteration of the endosomal trafficking pathways during the development of DC that parallels the changes in surface expression of MHCII is regulated at least in part by the activities of caspases, inducible NO synthetase, and its product NO.

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Figures

Fig. 1.

Fig. 1.

Expression of essential molecules of the endosomal pathways during DC maturation. Untreated (control) and LPS- or TNF-α-treated DC were extracted in buffer containing 2% Triton X-100 for 1 h on ice. Extracts (50 μg) were separated by 12% mini-SDS/PAGE and analyzed by Western blot by using Abs against α-adaptin, γ-adaptin, syntaxin (Syn) 7, syntaxin 8, dynamin (Dyn), Vti1A, and Vti1B. All of the Abs used in this experiment were mouse mAb, except for anti-syntaxin 7, which was a polyclonal Ab raised in a rabbit.

Fig. 2.

Fig. 2.

Effects of caspase inhibitors. (a) Inhibition of protein cleavage of endosomal trafficking molecules. DC were either treated with DMSO (control), group I caspase (CI) inhibitor, or group III caspase (CIII) inhibitor for 4 h before lysis and treatment with extraction buffer for 1 h on ice. Extracts (50 μg) were separated by 12% mini-SDS/PAGE and analyzed by Western blot by using Abs against α-adaptin, γ-adaptin, Vti1A, Vti1B, syntaxin (Syn) 7, syntaxin 8, and dynamin (Dyn) as probes. For Vti1B, syntaxin 7, and dynamin, only the DMSO- and group I caspase inhibitor-treated DC were analyzed. (b) Inhibition by LPS of cleavage of substrates for specific caspases. Extracts (20 μg) from untreated and LPS-treated DC were incubated with fluorescence-labeled substrates for caspase-1, caspase-2, caspase-3, and caspase-4 (see Materials and Methods) for 2, 4, and 6 h. The inhibition of caspase activity during DC maturation induced by LPS treatment is shown.

Fig. 4.

Fig. 4.

Caspase cleavage activities increased in iNOS-deficient DC. DC prepared from wild-type (wt) and iNOS-deficient (iNOS-/-) mice were either untreated or treated with LPS for 40 h before protein extract preparations. Protein extracts were analyzed by Western blot by using mAb against iNOS, α-adaptin, γ-adaptin, dynamin, syntaxin (Syn) 8, and Vti1A.

Fig. 6.

Fig. 6.

β- and γ-adaptin cleavage in maturing DC is different from that occurring during apoptosis. (a) BMDC, N9, and RAW cells were UV irradiated for 2 min to induce apoptosis. After 6 h of culture, a fraction of control or irradiated cells was stained with annexin V-FITC to ensure the irradiated population was in apoptosis. (b) Total cell lysate was prepared, and β- and γ-adaptin were analyzed by Western blot. As previously observed, only untreated BMDC present cleaved adaptin fragments. After UV irradiation, β- and γ-adaptin were cleaved in all cell types. The size of the cleaved fragment was different from the one previously observed in untreated BMDC, suggesting that different caspase pathways are active during DC maturation or during the apoptotic processes.

Fig. 3.

Fig. 3.

iNOS, induced by LPS, reduced the cleavage of α-adaptin and γ-adaptin during DC maturation. (a Upper) Protein extracts (80 μg) from immature (untreated) and mature DC (plus LPS) were analyzed by Western blot by employing mAb specific for iNOS (NOS2). (a Lower) Confocal microscopic analysis for iNOS (using anti-iNOS phycoerythrin) performed on immature and LPS-matured DC as above. The red dye appears white in this black and white figure. (b) Extracts from DC either untreated (lane 1) or treated with the iNOS inhibitor

l

-NMMA (lane 2), LPS (lane 3), or

l

-NMMA plus LPS (lane 4) were analyzed by Western blot by using Abs against α-adaptin.

Fig. 5.

Fig. 5.

DC treatment with group I caspase inhibitor (CI) as well as LPS induces MHCII redistribution to the cell surface. Cell-surface staining of GM-CSF, SJL/J BMDC untreated or treated with LPS or group I caspase inhibitor with the mAbs specific for empty (KL-304) or peptide-loaded (Y3P) MHCII. A typical experiment is shown.

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