Christiane Habich

Adaptive immunity in obesity and insulin resistance

Obesity is the hallmark of the metabolic syndrome and predisposes patients to the development of major chronic metabolic diseases including type 2 diabetes mellitus. Adipose tissue expansion in obesity is characterized by increasing infiltration of proinflammatory immune cells into adipose tissue causing chronic, low-grade inflammation. Phenotypic switching of macrophages is an important mechanism of adipose tissue inflammation, and there is involvement of cells from the adaptive immune system in this process. T-cell phenotype changes and recruitment of B cells and T cells precedes macrophage infiltration. Cytokines and chemokines produced by immune cells influence localized and systemic inflammation, which is a pathogenic link between obesity and insulin resistance. Antigens absorbed from the gut might contribute to T-cell activation and recruitment into visceral adipose tissue in obesity. This Review summarizes, in the context of obesity, the evidence for infiltration of adipose tissue by cells of the adaptive immune system, how adaptive system cells affect innate cell populations and the influence of adaptive immune cells on the development of insulin resistance.

The receptor for heat shock protein 60 on macrophages is saturable, specific, and distinct from receptors for other heat shock proteins.

Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4°C was followed by endocytosis at 37°C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the α2-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.

Heat shock protein 60: specific binding of lipopolysaccharide.

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [3H]LPS. [3H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335–366 of HSP60. Clone 4B9/89 also strongly inhibited [3H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331–380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351–370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354–365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.

Wheat Gluten Causes Dendritic Cell Maturation and Chemokine Secretion

Wheat gluten causes gut inflammation in genetically predisposed individuals. We tested the hypothesis that wheat gluten is not only a target of adaptive immunity, but also modulates the function of APC. Dendritic cells (DC) derived from the bone marrow of BALB/c mice were exposed to chymotrypsin-treated wheat gluten. This induced DC maturation as estimated by all surface markers tested (MHC class II, CD40, CD54, and CD86). The effect was dose dependent, and, at 100 μg/ml gluten matched that caused by 10 ng/ml LPS. A role of endotoxin contamination was ruled out by demonstrating the resistance of wheat gluten effects to LPS antagonist polymyxin B. DC from LPS nonresponder strain C3H/HeJ were affected by wheat gluten, but not by LPS. Proteinase K-digested wheat gluten was unable to stimulate DC maturation. Wheat gluten induced a unique secretion pattern of selected cytokines and chemokines in DC. Classic pro- or anti-inflammatory mediators were not produced, in contrast to LPS. Rather, chemokines MIP-2 and keratinocyte-derived cytokine were secreted in large amounts. We conclude that wheat gluten lowers the threshold for immune responses by causing maturation of APC, by attracting leukocytes and increasing their reactivity state. In the presence of an appropriate genetic predisposition, this is expected to increase the risk of adverse immune reactions to wheat gluten or to other Ags presented.

Different heat shock protein 60 species share pro-inflammatory activity but not binding sites on macrophages

In a study of seven different hsp60 species, we found that all mammalian and microbial proteins shared the property of eliciting an inflammatory response in mouse macrophages. In all cases, TNFα production was induced by 0.1 μM concentrations of hsp60. However, the different hsp60 preparations did not compete for the same binding site. The binding of fluorescence‐labeled human hsp60 was inhibited by excess unlabeled human, rat or mouse hsp60, but not hamster, Escherichia coli, Chlamydia pneumoniae or Mycobacterium bovis hsp60. We conclude that phylogenetically separate hsp60 species interact with innate immune cells via different recognition pathways.

Heat Shock Protein 60 as a Mediator of Adipose Tissue Inflammation and Insulin Resistance

The stress protein heat shock protein 60 (Hsp60) induces secretion of proinflammatory mediators from murine adipocytes. This study aimed to study Hsp60 as a mediator of adipose tissue inflammation and skeletal muscle cell (SkMC) insulin sensitivity and to quantify plasma Hsp60 concentrations in lean and obese individuals. Regulation of Hsp60 release and Hsp60-induced cytokine secretion and signaling was measured in human adipocytes and SkMCs. Adipocytes exhibited higher Hsp60 release than preadipocytes and SkMCs, which was further stimulated by cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated extracellular signal–related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK), p38, nuclear factor (NF)-κB, and impaired insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, Hsp60 stimulated adipocytes to secrete tumor necrosis factor-α, interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2, JNK, and NF-κB and inhibits insulin signaling and insulin-stimulated glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemoattractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher in obese males than in lean males and correlated positively with BMI, blood pressure, leptin, and homeostasis model assessment–insulin resistance. In summary, Hsp60 is released by human adipocytes, increased in plasma of obese humans, and induces insulin resistance. This is accompanied by activation of proinflammatory signaling in human adipocytes and SkMCs. Thus, Hsp60 might be a factor underlying adipose tissue inflammation and obesity-associated metabolic disorders.

Heat shock protein 60: Identification of specific epitopes for binding to primary macrophages

In the present study, we characterized regions of human heat shock protein (HSP) 60 responsible for binding to primary macrophages. Studies using 20‐mer peptides of the HSP60 sequence to compete with HSP60‐binding to macrophages from C57BL/6J mice showed that regions aa241–260, aa391–410 and aa461–480 are involved in surface‐binding. HSP60 mutants, lacking the N‐terminal 137, 243 or 359 amino acids, inhibited HSP60‐binding to primary macrophages to different degrees, demonstrating that all three regions are required for optimal binding. Analysis of different pro‐ and eukaryotic HSP60 species indicated that phylogenetically separate HSP60 species use different binding sites on primary macrophages.

Heat shock protein 60 induces inflammatory mediators in mouse adipocytes

Adipocytes represent an important cellular source of inflammatory mediators. However, the signals for the induction of proinflammatory adipocyte activities are largely unknown. Here, we demonstrate that heat shock protein (Hsp) 60, a potent stimulator of innate immunity, induces the release of the inflammatory mediators interleukin‐6, CXCL1 and monocyte chemoattractant protein‐1 in a time‐ and concentration‐dependent manner from cells of the adipocyte line 3T3‐L1 and from adipocytes of obese mice. These results identify Hsp60 as an important regulator of adipocyte functions which contribute to the development of inflammatory processes as observed in diabetes and diabetes‐associated complications.

Identification of the heat shock protein 60 epitope involved in receptor binding on macrophages

In the present study, we identified the human heat shock protein 60 (HSP60) epitope responsible for binding to macrophages. Studies using overlapping 15‐ and 20‐mer peptides of the human HSP60 sequence to compete with binding of HSP60 to macrophages indicated that surface binding was accounted for by the region aa481–500. Deletion mutants of HSP60, lacking the N‐terminal 137, 243 or 359 amino acids, strongly inhibited HSP60 binding to macrophages. Monoclonal antibodies addressing regions aa1–200, aa335–366 or aa383–447 did not block HSP60 binding. We conclude that a single C‐terminal region, aa481–500, accounts for the binding of HSP60 to macrophages.

Heat shock protein 60: evidence for receptor-mediated induction of proinflammatory mediators during adipocyte differentiation.

Adipocytes play important roles in lipid metabolism but also in the control of inflammatory processes. Based on our previous findings of heat shock protein (Hsp) 60‐induced activation of preadipocytes we investigated whether the capacity of heat shock protein 60 (Hsp60) to interact with adipocytes and to stimulate their proinflammatory activity is determined by the differentiation state of the cells. Hsp60 bound to adipocytes and stimulated the release of inflammatory mediators independent of their differentiation state. Hsp60‐adipocyte interactions revealed basic characteristics of a receptor‐mediated process. Our findings characterize Hsp60 binding and Hsp60‐induced release of proinflammatory mediators as fundamental properties of adipocytes independent of their differentiation state.