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A single amino acid determines the immunostimulatory activity of interleukin 10.

Ding Y, Qin L, Kotenko SV, Pestka S, Bromberg JS - J. Exp. Med. (2000)

Bottom Line: This single conservative residue alteration significantly affects ligand affinity for receptor; however, affinity changes do not necessarily alter specific activities for biologic responses in a predictable fashion.These results suggest complex regulation of IL-10 receptor-ligand interactions and subsequent biological responses.These results demonstrate that vIL-10 may represent a captured and selectively mutated cIL-10 gene that benefits viral pathogenesis by leading to ineffective host immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Cellular interleukin 10s (cIL-10s) of human and murine origin have extensive sequence and structural homology to the Epstein-Barr virus BCRF-I gene product, known as viral IL-10 (vIL-10). Although these cytokines share many immunosuppressive properties, vIL-10 lacks several of the immunostimulatory activities of cIL-10 on certain cell types. The molecular and cellular bases for this dichotomy are not currently defined. Here, we show that the single amino acid isoleucine at position 87 of cIL-10 is required for its immunostimulatory function. Substitution of isoleucine in cIL-10 with alanine, which corresponds to the vIL-10 residue, abrogates immunostimulatory activity for thymocytes, mast cells, and alloantigenic responses while preserving immunosuppressive activity for inhibition of interferon gamma production and prolongation of cardiac allograft survival. Conversely, substitution of alanine with isoleucine in vIL-10 converts it to a cIL-10-like molecule with immunostimulatory activity. This single conservative residue alteration significantly affects ligand affinity for receptor; however, affinity changes do not necessarily alter specific activities for biologic responses in a predictable fashion. These results suggest complex regulation of IL-10 receptor-ligand interactions and subsequent biological responses. These results demonstrate that vIL-10 may represent a captured and selectively mutated cIL-10 gene that benefits viral pathogenesis by leading to ineffective host immune responses. The ability to manipulate the activity of IL-10 in either a stimulatory or suppressive direction may be of practical value for regulating immune responses for disease therapy, and of theoretical value for determining what aspects of IL-10 activity are important for normal T cell responses.

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(a) Induction of MHC class I antigen expression by IL-10. The 16-9 CHO cell lines expressing an HLA-B7 reporter construct and hIL-10R1/hIFN-γR1 chimera and/or hIL-10R2 were exposed to control or COS cell supernatants (10%) containing hIL-10 or hIL-10(I87A) for 72 h and stained for HLA-B7. Open histograms represent negative controls; shaded histograms represent treated groups. (b) Induction of Stat1 activation by IL-10. The 16-9 cells expressing hIL-10R1/hIFN-γR1 chimera plus hIL-10R2 were exposed to COS supernatants (100%) for 15 min, the cells were lysed, and nuclear extracts were obtained for binding to 32P-labeled 22-bp IFN-γ activation sequence (GAS) from human IFN-γ regulatory factor 1 (hIRF-1). Only anti-Stat1 but not anti-Stat3 caused supershifting of the complex. (c) Induction of Stat1, Stat3, and Stat5 activation by IL-10. Ba/F3-mIL-10R1 cells were exposed to COS supernatants (100%) for 15 min, and the EMSAs were performed using a 32P-labeled 22-bp IFN-γ response region (GRR) probe.
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Figure 4: (a) Induction of MHC class I antigen expression by IL-10. The 16-9 CHO cell lines expressing an HLA-B7 reporter construct and hIL-10R1/hIFN-γR1 chimera and/or hIL-10R2 were exposed to control or COS cell supernatants (10%) containing hIL-10 or hIL-10(I87A) for 72 h and stained for HLA-B7. Open histograms represent negative controls; shaded histograms represent treated groups. (b) Induction of Stat1 activation by IL-10. The 16-9 cells expressing hIL-10R1/hIFN-γR1 chimera plus hIL-10R2 were exposed to COS supernatants (100%) for 15 min, the cells were lysed, and nuclear extracts were obtained for binding to 32P-labeled 22-bp IFN-γ activation sequence (GAS) from human IFN-γ regulatory factor 1 (hIRF-1). Only anti-Stat1 but not anti-Stat3 caused supershifting of the complex. (c) Induction of Stat1, Stat3, and Stat5 activation by IL-10. Ba/F3-mIL-10R1 cells were exposed to COS supernatants (100%) for 15 min, and the EMSAs were performed using a 32P-labeled 22-bp IFN-γ response region (GRR) probe.

Mentions: The activities of IL-10 are mediated through a cell surface receptor complex consisting of a ligand binding chain (IL-10R1) and a signal transducing accessory chain (IL-10R2) 333940. Although IL-10R2 alone does not bind IL-10, only cells expressing both IL-10R1 and IL-10R2 can mediate IL-10 signal transduction 33. To determine if the single amino acid change affected the receptor binding specificity of IL-10, we stimulated 16-9 CHO cells with IL-10. These cells had been stably transfected to express an HLA-B7 reporter gene along with hIL-10R2 alone; the hIL-10R1 chimeric receptor, in which the transmembrane and intracellular domains of the hIFN-γR1 chain were substituted for the transmembrane and intracellular domains of the hIL-10R1 chain; or both receptors. We previously showed that hIL-10 stimulates IFN-γ–like responses only in those cells that express both receptors, inducing both MHC class I antigen expression and Stat1 activation 33. Fig. 4 a shows that both hIL-10 and hIL-10(I87A) induced identical MHC class I expression only in cells expressing both receptors, demonstrating that both hIL-10 and hIL-10(I87A) require IL-10R1 and IL-10R2 to mediate signal transduction, and that the single amino acid change did not alter peptide receptor binding specificity. An EMSA was performed with the same cells to determine STAT activation. The results show the comparable formation of DNA-binding complexes in both hIL-10– and hIL-10(I87A)–treated cells, and these complexes were supershifted with anti-Stat1 but not anti-Stat3 antibodies (Fig. 4 b). To explore if this result can be extended to more physiological IL-10–responsive murine cells, the IL-3–dependent murine pro-B cell Ba/F3 line expressing mIL-10R1 was also used. This line proliferates in a concordant fashion to both cIL-10 and vIL-10 293031, and it was shown previously that Stat1, Stat3, and Stat5 are activated upon cIL-10 treatment in Ba/F3-mIL-10R1 cells 34. Fig. 4 c shows that both hIL-10 and hIL-10(I87A) induced Stat1, Stat3, and Stat5 activation. These results further demonstrate that the alanine substitution did not alter the receptor binding specificity and Janus kinase (JAK)-STAT signal transduction pathway activation in these particular cell lines that respond in a concordant fashion to cIL-10 and vIL-10.


A single amino acid determines the immunostimulatory activity of interleukin 10.

Ding Y, Qin L, Kotenko SV, Pestka S, Bromberg JS - J. Exp. Med. (2000)

(a) Induction of MHC class I antigen expression by IL-10. The 16-9 CHO cell lines expressing an HLA-B7 reporter construct and hIL-10R1/hIFN-γR1 chimera and/or hIL-10R2 were exposed to control or COS cell supernatants (10%) containing hIL-10 or hIL-10(I87A) for 72 h and stained for HLA-B7. Open histograms represent negative controls; shaded histograms represent treated groups. (b) Induction of Stat1 activation by IL-10. The 16-9 cells expressing hIL-10R1/hIFN-γR1 chimera plus hIL-10R2 were exposed to COS supernatants (100%) for 15 min, the cells were lysed, and nuclear extracts were obtained for binding to 32P-labeled 22-bp IFN-γ activation sequence (GAS) from human IFN-γ regulatory factor 1 (hIRF-1). Only anti-Stat1 but not anti-Stat3 caused supershifting of the complex. (c) Induction of Stat1, Stat3, and Stat5 activation by IL-10. Ba/F3-mIL-10R1 cells were exposed to COS supernatants (100%) for 15 min, and the EMSAs were performed using a 32P-labeled 22-bp IFN-γ response region (GRR) probe.
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Related In: Results  -  Collection

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Figure 4: (a) Induction of MHC class I antigen expression by IL-10. The 16-9 CHO cell lines expressing an HLA-B7 reporter construct and hIL-10R1/hIFN-γR1 chimera and/or hIL-10R2 were exposed to control or COS cell supernatants (10%) containing hIL-10 or hIL-10(I87A) for 72 h and stained for HLA-B7. Open histograms represent negative controls; shaded histograms represent treated groups. (b) Induction of Stat1 activation by IL-10. The 16-9 cells expressing hIL-10R1/hIFN-γR1 chimera plus hIL-10R2 were exposed to COS supernatants (100%) for 15 min, the cells were lysed, and nuclear extracts were obtained for binding to 32P-labeled 22-bp IFN-γ activation sequence (GAS) from human IFN-γ regulatory factor 1 (hIRF-1). Only anti-Stat1 but not anti-Stat3 caused supershifting of the complex. (c) Induction of Stat1, Stat3, and Stat5 activation by IL-10. Ba/F3-mIL-10R1 cells were exposed to COS supernatants (100%) for 15 min, and the EMSAs were performed using a 32P-labeled 22-bp IFN-γ response region (GRR) probe.
Mentions: The activities of IL-10 are mediated through a cell surface receptor complex consisting of a ligand binding chain (IL-10R1) and a signal transducing accessory chain (IL-10R2) 333940. Although IL-10R2 alone does not bind IL-10, only cells expressing both IL-10R1 and IL-10R2 can mediate IL-10 signal transduction 33. To determine if the single amino acid change affected the receptor binding specificity of IL-10, we stimulated 16-9 CHO cells with IL-10. These cells had been stably transfected to express an HLA-B7 reporter gene along with hIL-10R2 alone; the hIL-10R1 chimeric receptor, in which the transmembrane and intracellular domains of the hIFN-γR1 chain were substituted for the transmembrane and intracellular domains of the hIL-10R1 chain; or both receptors. We previously showed that hIL-10 stimulates IFN-γ–like responses only in those cells that express both receptors, inducing both MHC class I antigen expression and Stat1 activation 33. Fig. 4 a shows that both hIL-10 and hIL-10(I87A) induced identical MHC class I expression only in cells expressing both receptors, demonstrating that both hIL-10 and hIL-10(I87A) require IL-10R1 and IL-10R2 to mediate signal transduction, and that the single amino acid change did not alter peptide receptor binding specificity. An EMSA was performed with the same cells to determine STAT activation. The results show the comparable formation of DNA-binding complexes in both hIL-10– and hIL-10(I87A)–treated cells, and these complexes were supershifted with anti-Stat1 but not anti-Stat3 antibodies (Fig. 4 b). To explore if this result can be extended to more physiological IL-10–responsive murine cells, the IL-3–dependent murine pro-B cell Ba/F3 line expressing mIL-10R1 was also used. This line proliferates in a concordant fashion to both cIL-10 and vIL-10 293031, and it was shown previously that Stat1, Stat3, and Stat5 are activated upon cIL-10 treatment in Ba/F3-mIL-10R1 cells 34. Fig. 4 c shows that both hIL-10 and hIL-10(I87A) induced Stat1, Stat3, and Stat5 activation. These results further demonstrate that the alanine substitution did not alter the receptor binding specificity and Janus kinase (JAK)-STAT signal transduction pathway activation in these particular cell lines that respond in a concordant fashion to cIL-10 and vIL-10.

Bottom Line: This single conservative residue alteration significantly affects ligand affinity for receptor; however, affinity changes do not necessarily alter specific activities for biologic responses in a predictable fashion.These results suggest complex regulation of IL-10 receptor-ligand interactions and subsequent biological responses.These results demonstrate that vIL-10 may represent a captured and selectively mutated cIL-10 gene that benefits viral pathogenesis by leading to ineffective host immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Cellular interleukin 10s (cIL-10s) of human and murine origin have extensive sequence and structural homology to the Epstein-Barr virus BCRF-I gene product, known as viral IL-10 (vIL-10). Although these cytokines share many immunosuppressive properties, vIL-10 lacks several of the immunostimulatory activities of cIL-10 on certain cell types. The molecular and cellular bases for this dichotomy are not currently defined. Here, we show that the single amino acid isoleucine at position 87 of cIL-10 is required for its immunostimulatory function. Substitution of isoleucine in cIL-10 with alanine, which corresponds to the vIL-10 residue, abrogates immunostimulatory activity for thymocytes, mast cells, and alloantigenic responses while preserving immunosuppressive activity for inhibition of interferon gamma production and prolongation of cardiac allograft survival. Conversely, substitution of alanine with isoleucine in vIL-10 converts it to a cIL-10-like molecule with immunostimulatory activity. This single conservative residue alteration significantly affects ligand affinity for receptor; however, affinity changes do not necessarily alter specific activities for biologic responses in a predictable fashion. These results suggest complex regulation of IL-10 receptor-ligand interactions and subsequent biological responses. These results demonstrate that vIL-10 may represent a captured and selectively mutated cIL-10 gene that benefits viral pathogenesis by leading to ineffective host immune responses. The ability to manipulate the activity of IL-10 in either a stimulatory or suppressive direction may be of practical value for regulating immune responses for disease therapy, and of theoretical value for determining what aspects of IL-10 activity are important for normal T cell responses.

Show MeSH
Related in: MedlinePlus