Alteration of HLA-B27 peptide presentation after infection of transfected murine L cells by Shigella flexneri

Abstract

Shigella flexneri is a triggering agent for reactive arthritis in HLA-B27-susceptible individuals. Considering the intracellular multiplication of bacteria, it seems likely that bacterial peptides may be presented by the major histocompatibility complex (MHC) class I pathway. To examine this hypothesis, we infected HLA-B*2705- and/or human beta2-microglobulin-transfected murine L-cell lines with M90T, an invasive strain of S. flexneri. Bacterial infection induced no detectable modifications in the biosynthesis and expression level of HLA-B27, as assessed by immunoprecipitation, Northern blot analysis, and flow cytometry. Using confocal microscopy, we observed that bacterial infection induced a clustering of HLA-B27 molecules during macropinocytosis and before bacterial dissemination from cell to cell. Peptides naturally bound to HLA-B27 molecules were acid eluted from infected cells and separated by high-performance liquid chromatography. Major differences were observed in high-performance liquid chromatography profiles and in the nature of peptides presented following bacterial infection. Although most of the antigens presented were not accessed by Edman degradation, we obtained two sequences partially homologous to bacterial proteins. These peptides lacked the major HLA-B27 peptide anchor (Arg) at position 2, and one had an unusual length of 14 amino acids. These data suggest that alterations in the peptide presentation by HLA-B27 occur during infection, which could be relevant to the pathogenesis of HLA-B27-related arthritis.

FIG. 1

Immunoprecipitation of HLA-B27 molecules from noninfected and infected JT1 cells. JT1 cells were infected either with the invasive strain S. flexneri M90T or with the noninvasive strain BS176. Sixty minutes after infection, cells were washed and incubated for a further 60 min with [³⁵S]methionine; 2.8 × 10⁶ cells were lysed, and the lysate was precleared before immunoprecipitation using MAb B1.23.2, specific for HLA-B,C. The gel was loaded in duplicate.

FIG. 2

Northern blot analysis of HLA-B27 mRNA in infected JT1 cells. JT1 cells were either mock infected or infected with the invasive strain S. flexneri M90T. At indicated times postinfection, total RNA was extracted, separated by electrophoresis on an agarose-formaldehyde gel, and transferred to a membrane. The membrane was sequentially hybridized with a 0.5-kb HLA-B7 probe which recognizes HLA-B27 RNA and then with a GAPDH probe. The cell line 7.3.13, expressing the human β2m only, was used as a control. The HLA-B27 transcript is designated by an arrow.

FIG. 3

FACS analysis of total HLA-B27 expression in JT1 cells. JT1 cells were either mock infected (A) or infected with the invasive strain S. flexneri M90T for 2.5 h (B) and permeabilized before labeling with MAbs B1.23.2, ME1, and HC10. Infection was controlled by using a polyclonal antibody (FlexV) raised against S. flexneri serotype 5 (C). Specific staining in noninfected and infected cells is represented by light and bold lines, respectively; nonspecific staining is represented by dotted lines. Fluorescence intensity is expressed on the FL1 x axis, and the relative number of cells is shown on the y axis.

FIG. 4

Confocal fluorescence analysis of HLA-B27 molecules in JT1 cells infected with S. flexneri M90T. JT1 cells were either mock infected (A) or infected with the invasive strain M90T (B to D) and were fixed in paraformaldehyde (3.7% in PBS) at 15 min postinfection (B) or at 2.5 h postinfection and permeabilized (C and D). Cells were stained first with MAb B1.23.2 (A to C) or HC10 (D) coupled to FITC-conjugated GAMIg and then with FlexV coupled to Texas red-conjugated goat anti-rabbit immunoglobulins. Nuclei were stained with DAPI (A and C).

FIG. 5

HPLC profiles of peptides eluted from HLA-B27 molecules purified from uninfected (A) or S. flexneri M90T-infected (B) JT1 cells. The separation was performed on a C₁₈ column with an increasing gradient of acetonitrile in 0.05% TFA. Fractions sequenced by Edman NH₂-terminal degradation are indicated by arrows.