Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes - PubMed (original) (raw)

Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes

C Forestier et al. Infect Immun. 1999 Aug.

Abstract

Lipopolysaccharide (LPS), a major amphiphilic molecule located at the outer membrane of gram-negative bacteria, is a potent antigen known to induce specific humoral immune responses in infected mammals. LPS has been described as a polyclonal activator of B lymphocytes, triggering the secretion of antibodies directed against distinct sugar epitopes of the LPS chain. But, how LPS is handled by B cells remains to be fully understood. This task appears to be essential for a better knowledge of the anti-LPS humoral immune response. In this study, we examine the internalization of LPS and its interaction with antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human B-cell lines. By use of immunofluorescence, we observe that structurally different LPSs from Brucella and Shigella strains accumulate in an intracellular compartment enriched in MHC class II molecules. By use of immunoprecipitation, we illustrate that only Brucella abortus LPS associates with MHC class II molecules in a haplotype-independent manner. Taken together, these results raise the possibility that B. abortus LPS may play a role in T-cell activation.

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Figures

FIG. 1

B. abortus LPS accumulates in lysosomal compartments. Human B lymphocytes were incubated for 4 h at 37°C with B. abortus LPS. After fixation and permeabilization, double immunofluorescence experiments were analyzed by confocal microscopy. B. abortus LPS (A) was found to colocalize with the lysosomal marker cathepsin D (B). Optical sections of 0.5 μm are presented. Bar, 13 μm.

FIG. 2

LPS heterogeneity is conserved after internalization. A total of 0.3 μg of native B. abortus (A, lane b), B. melitensis (A, lane c), or S. flexneri LPS (B, lane a) were immunoprecipitated, and immunoblots were revealed by using the Baps3C/Y anti-Brucella O-chain antibody (A) and the IgAC5 anti-Shigella O chain (B). In parallel, human B lymphocytes were incubated for 4 h at 37°C with B. abortus (A, lane d), B. melitensis (A, lane c), or S. flexneri LPS (B, lane b). After cell lysis, LPSs were immunoprecipitated, and immunoblots were revealed by use of the respective specific antibodies. In control experiments (A, lane a, and B, lane c), immunoprecipitations were performed in the absence of LPS. The asterisk indicates the light chain of IgG used for immunoprecipitation as revealed by the secondary antibody.

FIG. 3

LPS concentrates in MHC class II-enriched intracellular compartments. Human AT24B B lymphocytes were incubated for 4 h at 37°C with B. abortus LPS. After fixation and permeabilization, double immunofluorescence experiments were analyzed by confocal microscopy. (A) LPS was stained by a rabbit serum anti-LPS followed by a Texas red-conjugated anti-rabbit antibody. (B) HLA-DR molecules recognized by an anti-HLA-DR mouse monoclonal antibody were revealed by use of a donkey anti-mouse IgG coupled to fluorescein. Bar, 10 μm.

FIG. 4

B. abortus LPS associates with murine Iak molecules. Murine 2A4 cells were incubated with B. abortus LPS (A, lanes b and c, and B, lanes a and b) or with cell culture medium alone (A, lane a) for 4 h at 37°C. Cells were lysed, and immunoprecipitations were processed by using the 10.2.16 anti-class II antibody (lanes a and b) or the control IgG2b isotype antibody (lane c) in panel (A) and the Baps C/Y anti-Brucella O-chain antibody (lane b) and the control IgG3 isotype antibody (lane a) in panel B. Immunoblots were revealed by using the mouse anti-O-chain antibody followed by a peroxidase-conjugated goat anti-mouse IgG antibody in panel A and the rabbit anti-MHC class II Iaβ-chain antibody followed by a peroxidase-conjugated goat anti-rabbit IgG antibody in panel B.

FIG. 5

B. abortus LPS coprecipitates with MHC class II molecules. (A) 2A4 B cells were incubated with B. melitensis (lanes a and b), B. abortus (lanes c and d), or S. flexneri (lanes e and f) LPS. Class II molecules were precipitated with the 10.2.16 anti-IAk (lanes a, c, and e) antibody, and class I molecules were precipitated with the anti-H2k antibody (lanes b, d, and f). (B) Murine LK B cells were incubated for 4 h at 37°C with B. abortus (lane a) or B. melitensis (lane b) LPS, and class II molecules were immunoprecipitated with the M5114 antibody. (C) Human AT24B B lymphocytes were incubated for 4 h at 37°C with B. abortus (lane a), B. melitensis (lane b), or S. flexneri (lane c) LPSs. After cell lysis, HLA-DR molecules were precipitated. Detection of class II molecule-associated LPS was performed as described for Fig. 2. The asterisk indicates the light chain of IgG used for immunoprecipitation of the class II molecules in panels B and C as well as the heavy chain of IgG in panel B as revealed by the secondary antibody.

FIG. 6

Chemical models of LPS (A), B. abortus 2308 (B), B. melitensis 16M (C), and S. flexneri 5a (D) O chains. The B. abortus O chain adopts a rod-like structure with a cross-section of ca. 10 Å, with a distance of 20 Å separating the first and sixth formamido groups. The presence of an α1,3 linkage at every fifth residue in the B. melitensis O chain introduces a serious kink into the rod-like structure (showed by an arrow in panel C).

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Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes - PubMed (original) (raw)