Regulation of peptide presentation by major histocompatibility complex class II molecules at the surface of macrophages

Delwig, Alexei von; Musson, Julie A.; McKie, Norman; Gray, Joe; Robinson, John H.

doi:10.1002/eji.200324461

Cited by 7 publications

(18 citation statements)

References 56 publications

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“…To test antigen presentation of CII digests, 40-l aliquots of each digest were added to fixed THP-1 cells in the presence of the same doses of the enzyme inhibitors PMSF, E-64d, pepstatin, and phenanthroline to prevent further degradation (35) and then incubated for 18 hours at 37°C. To localize peptide/DR1 complexes in subcellular fractions, vesicular compartments present in the Percoll gradient fractions were added to 96-well plates and tested with T cell hybridomas.…”

Section: Methodsmentioning

confidence: 99%

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

Delwig

Altmann

Isaacs

et al. 2006

Arthritis & Rheumatism

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Objective. Type II collagen (CII) is a candidate autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of CII could alter intracellular antigen processing, leading to the development of autoimmune T cell responses. To address this possibility, we studied the intracellular processing of CII for presentation of the arthritogenic glycosylated epitope CII 259-273 to CD4 T cells in macrophages from HLA-DR1-transgenic mice.Methods. HLA-DR1-transgenic mice were generated on a class II major histocompatibility complexdeficient background, and T cell hybridomas specific for the glycosylated and nonglycosylated epitope CII [259][260][261][262][263][264][265][266][267][268][269][270][271][272][273] were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype of proteinases involved.Results. We showed that the glycosylated CII 259-273epitope required more extensive processing than did the nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in the processing of CII for antigen presentation, since these compartments contained 1) enzyme activity that generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide-receptive HLA-DR1 molecules, and 4) peptide/HLA-DR1 complexes that could directly activate T cell hybridomas. Degradation of CII by dense fractions occurred optimally at pH 4.5 and was abrogated by inhibitors of serine and cysteine proteinases. Conclusion. Processing of the arthritogenic glycosylated CII259-273 epitope, which is implicated in the induction of autoimmune arthritis, is more stringently regulated than is processing of the nonglycosylated form of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel therapeutic targets for the treatment of RA.

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Section: Methodsmentioning

confidence: 99%

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

Delwig

Altmann

Isaacs

et al. 2006

Arthritis & Rheumatism

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“…Purified MHC‐II molecules have been shown to bind peptides containing T‐cell epitopes and partially to protect them from degradation by exogenously added proteolytic enzymes, such as cathepsin B, pronase E and chymotrypsin, for presentation to CD4 T cells in cell‐free systems, 20,21 although the factors influencing the rescue of exogenous peptides by MHC‐II expressed at the cell‐surface of APC for presentation to CD4 T cells are not well understood. In our previous study we showed that enzymes of the metallo‐, aspartic‐ and serine‐proteinase families are involved in destructive processing of exogenous peptides at the surface of macrophages 22 . In this report, we show that binding to MHC‐II molecules at the surface of APC may protect a significant proportion of exogenous peptides from proteolytic degradation.…”

Section: Introductionmentioning

confidence: 56%

“…Macrophages were fixed with 1·0% paraformaldehyde (w/v in Hanks' balanced salt solution; HBSS) for 4 min to block antigen internalization. This treatment has been previously shown to preserve cell surface‐associated enzyme activity without exposing lysosomal enzymes 17,22 . To test the effect of pH on peptide presentation, HBSS pH 7·2; and 0·1 m sodium citrate buffers with pH 6·5 and pH 5·5 were used.…”

Section: Methodsmentioning

confidence: 99%

Major histocompatibility class II molecules prevent destructive processing of exogenous peptides at the cell surface of macrophages for presentation to CD4 T cells

Delwig¹,

Musson²,

Gray

et al. 2005

Immunology

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Summary We studied factors affecting major histocompatibility complex class II (MHC‐II)‐restricted presentation of exogenous peptides at the surface of macrophages. We have previously shown that peptide presentation is modulated by surface‐associated proteolytic enzymes, and in this report the role of the binding of MHC‐II molecules in preventing proteolysis of exogenous synthetic peptides was addressed. Two peptides containing CD4 T‐cell epitopes were incubated with fixed macrophages expressing binding and non‐binding MHC‐II, and supernatants were analysed by high‐performance liquid chromatography and mass spectrometry to monitor peptide degradation. The proportion of full‐length peptides that were degraded and the number of peptide fragments increased when non‐binding macrophages were used, leading to reduction in peptide presentation. When MHC‐II molecules expressed on the surface of fixed macrophages were blocked with monoclonal antibody and incubated with peptides and the supernatants were transferred to fixed macrophages, a significant reduction in peptide presentation was observed. Peptide presentation was up‐regulated at pH 5·5 compared to neutral pH, and the latter was found to be the pH optimum of the proteolytic activity of the surface enzymes involved in the degradation of exogenous peptides and proteins. The data suggest that MHC‐II alleles that bind peptides protect them from degradation at the antigen‐presenting cell surface for presentation to CD4 T cells and we argue that this mechanism could be particularly pronounced at sites of inflammation.

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“…At the end of the incubation time, 30 ml aliquots of the reaction mixture were transferred to 96-well plates containing 4 Â 10 4 /well prefixed macrophages in 0.1 M Na-citrate buffer (pH 5.5) supplemented with the following broad-spectrum enzyme inhibitors to block further processing [21]: pepstatin A (inhibitor of aspartic proteinases, 0.5 mM [22]), 1,10-phenanthroline (inhibitor of metalloproteinases, 0.1 mM [23]), phenylmethylsulfonyl fluoride (inhibitor of serine proteinases, 3.0 mM [24]) and E-64d (inhibitor of cysteine proteinases, 10 mM [25]). Following 18 h incubation, plates were washed to remove inhibitors and digests, and specific T-cell hybridomas were added in 200 ml culture medium (4 Â 10 4 /well).…”

Section: After Incubation At 37mentioning

confidence: 99%

“…Productive processing of peptide epitopes that directly load MHC-II molecules for presentation to CD4 T cells was assessed by incubation of digests with prefixed macrophages in the presence of enzyme inhibitors to prevent further degradation by surface enzymes [21]. rPA digested by two regions of the gradient (fractions 4-7 and 24-28) was presented to a T-cell hybridoma specific for the epitope PA 547À560 (Fig.…”

Section: Localization Of Antigen-processing Activity In Subcellular Fmentioning

confidence: 99%

Distribution of Productive Antigen‐Processing Activity for MHC class II Presentation in Macrophages

et al. 2005

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We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigenpresenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.

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Regulation of peptide presentation by major histocompatibility complex class II molecules at the surface of macrophages

Cited by 7 publications

References 56 publications

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

The impact of glycosylation on HLA–DR1–restricted T cell recognition of type II collagen in a mouse model

Major histocompatibility class II molecules prevent destructive processing of exogenous peptides at the cell surface of macrophages for presentation to CD4 T cells

Distribution of Productive Antigen‐Processing Activity for MHC class II Presentation in Macrophages

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