Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity?

Abstract

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.

Fig. 1

Reciprocal intermolecular spreading is observed only between Grp78 and Ro (SS-A) antigens. (a) Kinetics of autoantibody production in C3H/HeJ mice immunized with 100 µg Grp78 (a) or Hsp72 (b) in Freund's complete adjuvant (day 0) and boosted twice subcutaneously with 100 µg Grp78 (a) or Hsp72 (b) in Freund's incomplete adjuvant (days 14 and 28). Sera were taken at weekly intervals and assessed for reactivity towards the immunogen (Grp78 (□)), mRo52 (○), Hsp72 (◊), mRo60 (▪), mCR (♦), mla (•) and the control protein MBP (▵) by ELISA. Immunoblot of Grp78 (c) and Hsp72 (d) immune sera against recombinant autoantigens (1 µg/lane) electrophoresed on a 10% SDS-PAGE gel.

Fig. 2

Immunoblots of Grp78- and Hsp70-immunized mice with murine lysates reveal reactivity with endogenous HSPs. Murine EL-4 cell lysates were prepared after heat shock for 30 min at 45°C and electrophoresed in 10% SDS–PAGE. The lysates were probed with Grp78 and Hsp70 immune sera as well as with commercially available anti-Grp78 monoclonal and polyclonal antibodies and an anti-Hsp70 monoclonal antibody.

Fig. 3

Region of mRo52 associates with Grp78. A region of mRo52 (amino acids 378–391) was examined in a competition gel shift assay for the ability to dissociate Grp78 from a complex with a model substrate molecule [reduced and carboxy-methylated α-lactalbumin (RCMLA)]. Complexes of Grp78–RCMLA were incubated with graded concentrations of the competitor peptide (0–200 µm) for 2 h at 37°C and electrophoresed under native PAGE conditions. Grp78 was detected by immunoblot using a specific monoclonal antibody. (b) The intensity of bands in the immunoblot was assessed by laser scanning densitometry and the percentage competition plotted as a function of peptide concentration. An IC₅₀ of 60 µm was derived for the mRo52 378–391 peptide, indicative of moderate to strong binding. (c) A capture ELISA showing specific capture of Grp78 by a multimeric Ro52 MAP 378–391 peptide construct (♦) compared to an irrelevant MAP from La 25–44 () or 287–301 (not shown).

Fig. 4

Preliminary analysis of Grp78 surface binding to a population of murine splenocytes. Single cell suspensions of splenocytes prepared from the spleens of C3H/HeJ mice were incubated for 30 min with PBS (a) or 1 µg/ml of biotinylated Grp78 (b), Hsp70 (c) or Hsp90 (d). After three washes, the cells were incubated with streptavidin–phycoerythrin conjugate and cells examined by flow cytometry. Results are shown as PE fluorescence on the y-axis and forward scatter on the x-axis.

Fig. 5

Model of Grp78-mediated immunity. The intra- and extracellular pool of Grp78 can modulate immunity towards bound antigen. I. Under normal physiological conditions Grp78 is sequestered in the ER and does not become a target of CD4⁺ T cells. II. The high level of homology between Gp78 and pathogen-derived HSPs may fuel Grp78-mediated intermolecular help towards Ro-specific B cells. III. Grp78 interacts with viral and other pathogen-derived glycoproteins in the ER and becomes exposed to autoreactive environments. IV. Many of these infections are also associated with apoptosis of the infected cell. Grp78-Ro complexes may concentrate in apoptotic blebs and either the blebs taken up by directly by APC or the released complexes may be taken up by receptor mediated phagocytosis, facilitating the ensuing autoreactivity.