HMGB1 is an endogenous immune adjuvant released by necrotic cells

Abstract

Immune responses against pathogens require that microbial components promote the activation of antigen-presenting cells (APCs). Autoimmune diseases and graft rejections occur in the absence of pathogens; in these conditions, endogenous molecules, the so-called 'innate adjuvants', activate APCs. Necrotic cells contain and release innate adjuvants; necrotic cells also release high-mobility group B1 protein (HMGB1), an abundant and conserved constituent of vertebrate nuclei. Here, we show that necrotic HMGB1(-/-) cells have a reduced ability to activate APCs, and HMGB1 blockade reduces the activation induced by necrotic wild-type cell supernatants. In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines.

Figure 1

The supernatants of necrotic cells that cause dendritic cell maturation contain HMGB1. (A) The expression of the CD83 and CD86 surface molecules was assessed by flow cytometry on untreated immature human DCs or DCs challenged with freeze/thawed (F/T) HeLa and polymorphonuclear cells (PMNs) or with their supernatants (sup). Only DCs treated with necrotic HeLa cells or their supernatants had significantly higher expression of the markers (P<0.001). (B) The intracellular distribution of HMGB1 was assessed by immunohistochemistry in PMNs and HeLa cells. Nuclei were revealed by staining with DAPI. (C) HLA-DR, CD40, CD83 and CD86 surface molecules were assessed by flow cytometry on immature DCs untreated or treated with the supernatant of necrotic HeLa cells (F/T sup) in the absence or in the presence of anti-HMGB1 antibodies (Ab), of irrelevant control antibodies or the HMGB1 inhibitory fragment box A. Results are expressed as relative increase (y-axis) in surface expression. DCs treated with F/T sup and anti-HMGB1 antibodies had significantly lower expression of the markers (^*P<0.005). Experiments were repeated at least three times with DCs from different donors. (D) HMGB1 was assessed by western blotting in the pellet (P) or in the supernatant (S) of necrotic F/T HeLa or PMNs. As a control, purified recombinant HMGB1 (rHMGB1; 10, 50 or 100 ng/lane) was used.

Figure 2

HMGB1 is required for the in vivo adjuvant activity of necrotic cells. (A) HMGB1 is expressed in the nuclei of wild-type (+/+, bottom) fibroblasts, whereas Hmgb1^−/− fibroblasts (top) do not express it. Nuclei are revealed by staining with DAPI, and HMGB1 expression by immunohistochemistry. (B) HMGB1 in the supernatant of necrotic wild-type (+/+) or Hmgb1 knockout (−/−) fibroblasts was revealed by immunoblotting. (C) The expression of HLA-DR, CD40, CD83, CD80 and CD86 was assessed by flow cytometry on untreated immature DCs (a) or DCs treated with the supernatants of wild-type necrotic fibroblasts (F/T +/+; b) or their −/− counterparts (c). Only DCs treated with supernatants of necrotic Hmgb1^+/+ fibroblasts had significantly higher expression of the markers (P<0.005). (D) The expression of HLA-DR, CD80 and CD86 was assessed on immature DCs, either untreated or treated with the supernatants of necrotic Hmgb1^+/+ fibroblasts (F/T +/+ sup), insoluble fractions of necrotic Hmgb1^+/+ fibroblasts (F/T +/+ pellet), supernatants of necrotic Hmgb1^−/− fibroblasts (F/T −/− sup) and insoluble fractions of necrotic Hmgb1^−/− fibroblasts (F/T ^−/− pellet). Results are expressed as a relative increase (y-axis) in surface expression over untreated DCs. DCs treated with pellets or F/T −/− sup had significantly lower expression of the different markers (^*P<0.01). Experiments were repeated at least three times with DCs from different donors. (E) The production of TNF-α by immature DCs treated with F/T −/− sup or F/T −/− sup reconstituted with purified recombinant HMGB1 was assessed in the cell culture supernatants. (F) The development of lymphoma was evaluated in C57BL/6 mice vaccinated with PBS (saline), apoptotic RMA cells or apoptotic RMA cells in the presence of the supernatants of wild-type necrotic fibroblasts (F/T +/+) or from their Hmgb1^−/− counterparts (F/T −/−). The results shown depict the fraction of tumour-free mice (y-axis) at different times after injection of living RMA lymphoma cells (x-axis). Protected mice were re-challenged on day 80 (arrow). (G) The results shown depict the mean diameter of the growing tumour (y-axis) at different times after administration of living RMA cells (x-axis). Fisher's exact test results for protection and tumour growth were ^**P<0.005 and ^***P<0.001, respectively.

Figure 3

HMGB1 expression and purification. (A) His–HMGB1 was purified by affinity chromatography from total lysates (TL) of COS7 cells. TL was applied to pre-packed Ni²⁺ columns and fractions were analysed by SDS–PAGE (FT, flow-through). Immunoblotting with anti-His or anti-HMGB1 antibodies shows that only the His–HMGB1 protein is retained and released by competitive elution with imidazole. His–HMGB1 has a higher molecular weight than endogenous HMGB1 because of the His-tag insertion. (B,C) Recombinant His–HMGB1 and HMGB1 were pure, as shown by Coomassie staining of an SDS–polyacrylamide gel.

Figure 4

In vivo adjuvant activity of HMGB1 towards soluble and cell-associated antigens. (A) The production of OVAspecific IgG was evaluated in C57BL/6 mice vaccinated with PBS (saline), OVA, OVA plus HMGB1 or HMGB1 alone. The results shown are expressed as mean±s.e.m. of IgG end-point titres (n=5/group). (B) The production of OVA-specific IgG was evaluated in groups of C3H/HeN or C3H/HeJ mice vaccinated with PBS, OVA, OVA plus HMGB1, OVA plus His–HMGB1 or OVA+CFA. Results are expressed as mean±s.e.m. of IgG end-point titres (n=5/group). Differences were statistically significant (^*P<0.01 with respect to OVA-injected animals). (C) The development of lymphoma was evaluated in mice vaccinated with PBS, apoptotic RMA cells or apoptotic RMA cells in the presence of rHMGB1 (0.5 μg/mouse). Results depict the fraction of tumour-free mice (y-axis) at different times after injection of living RMA cells (x-axis). Protected mice were re-challenged with living RMA on day 80 (arrow). (D) The subcutaneous growth of RMA cells was evaluated in vaccinated C57BL/6 mice (see above). The results shown depict the mean diameter±s.d. of the growing tumour (y-axis) at different times after injection of living RMA cells (x-axis).