Gerhard J. Zlabinger

Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production

Objective:Obesity is associated with a chronic low-grade inflammation and an increased abundance of macrophages in adipose tissue. Adipose tissue macrophages (ATMs) are assumed to interfere with adipocyte function leading to insulin resistance, thereby contributing to the pathogenesis of type 2 diabetes mellitus. Macrophages exist in separate types of differentiation, but the nature of ATMs is largely unknown.Design and measurements:Stromal vascular cells (SVCs) and ATMs were isolated from human adipose tissues from different locations. We characterized ATMs phenotypically and functionally by flow cytometry, endocytosis assay and determination of secreted cytokines. For comparison, we used macrophages of the ‘classical’ (M1) and the ‘alternative’, anti-inflammatory (M2) type differentiated in vitro from peripheral blood monocytes.Results:Like prototypic M2 macrophages, ATMs expressed considerable amounts of mannose receptor, haemoglobin scavenger receptor CD163 and integrin αvβ5. The number of cells expressing these molecules correlated significantly with the donors’ body mass indices (BMIs). Notably, SVCs positive for the common monocyte/macrophage marker CD14 contained a considerable fraction of blood monocytes, the abundance of which did not correlate with the BMIs, pointing to the requirement of the surface markers identified here for the identification of ATMs. ATMs showed endocytic activities similar to M2 macrophages and accordingly secreted high amounts of IL-10 and IL-1 receptor antagonist. However, basal and induced secretion of pro-inflammatory mediators TNF-α, IL-6, IL-1, MCP-1 and MIP-1α was even higher in ATMs than in pro-inflammatory M1 macrophages.Conclusion:ATMs comprise a particular macrophage type that is M2-like by surface marker expression, but they are competent to produce extensive amounts of inflammatory cytokines, which could considerably contribute to the development of insulin resistance.

B7-H1 (Programmed Death-1 Ligand) on Dendritic Cells Is Involved in the Induction and Maintenance of T Cell Anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-α, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.

CC Chemokine and CC Chemokine Receptor Profiles in Visceral and Subcutaneous Adipose Tissue Are Altered in Human Obesity

BACKGROUND/AIMS Obesity is associated with a low-grade inflammation, insulin resistance, and macrophage infiltration of adipose tissue. The role of CC chemokines and their respective receptors in human adipose tissue inflammation remains to be determined. METHODS sc and visceral adipose tissue of obese patients (body mass index 53.1 +/- 11.3 kg/m(2)) compared with lean controls (body mass index 25.9 +/- 3.8 kg/m(2)) was analyzed for alterations in inflammatory gene expression. RESULTS Macrophage infiltration was increased in sc and visceral adipose tissue of obese patients as determined by increased mRNA expression of a macrophage-specific marker (CD68) and by elevated macrophage infiltration. Gene expression of CC chemokines involved in monocyte chemotaxis (CCL2, CCL3, CCL5, CCL7, CCL8, and CCL11) and their receptors (CCR1, CCR2, CCR3, and CCR5) was higher in sc and visceral adipose tissue of obese patients. Serum concentrations of the inflammatory marker IL-6 and C-reactive protein were elevated in obese patients compared with lean controls. Obese patients revealed increased insulin resistance as assessed by the homeostasis model assessment of insulin resistance index and reduced plasma adiponectin concentrations. Adipose tissue expression of many CC chemokines and their receptors in the obese group positively correlated with CD68 expression. CONCLUSION Up-regulation of the CC chemokines and their respective receptors in adipose tissue occurs in human obesity and is associated with increased systemic inflammation.

Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4–dependent mechanism

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-γ production.

Intraocular concentrations of growth factors and cytokines in retinal vein occlusion and the effect of therapy with bevacizumab.

PURPOSE To investigate concentrations of growth factors and inflammatory cytokines in eyes with central (CRVO) and branch (BRVO) retinal vein occlusion before and during therapy with bevacizumab and to identify associations with disease activity. METHODS In a prospective clinical trial, 13 eyes of patients with CRVO (n = 5) or BRVO (n = 8) were included. Bevacizumab was administered intravitreously at baseline and months 1 and 2. Retreatments were given at monthly visits if OCT showed edema or when vision loss occurred. Aqueous humor samples were taken each time injections were performed. Follow-up was 15 months. Samples from patients with cataract served as the control. Multiplex bead assays were used for measurement of 28 growth factors and cytokines. RESULTS During therapy with bevacizumab, VEGF levels were reduced to below detection in the first 2 months. Whenever criteria for retreatment were met, VEGF was measurable again. The decrease in VEGF was associated with a decrease in central retinal thickness (CRT) and improvement in visual acuity (VA). Significantly increased concentrations of VEGF, IL-6, IL-8, IP-10, MCP-1, and PDGF-AA were observed in aqueous humor samples of patients with CRVO compared with the control samples. CONCLUSIONS VEGF levels were significantly elevated in patients with CRVO compared with control subjects. Intravitreal injections of bevacizumab resulted in a substantial decrease of VEGF under physiologic levels and remained low under the loading dose of three consecutive monthly retreatments. Macular edema was related to VEGF levels in the aqueous humor.

A Versatile Role of Mammalian Target of Rapamycin in Human Dendritic Cell Function and Differentiation

The mammalian target of rapamycin (mTOR) regulates cell growth and survival and exists as rapamycin-sensitive mTOR complex (mTORC) 1 and as rapamycin-insensitive mTORC2. Although mTOR is a well-known regulator of diverse immune cells, its detailed role in human dendritic cell (DC) function and differentiation is only incompletely understood. In this study, we demonstrate divergent roles of mTOR during activation and differentiation of myeloid DCs (mDCs) and monocyte-derived DCs (moDCs). Inhibition of mTORC1 in mDCs activated with TLR-dependent or -independent stimuli increased proinflammatory cytokines and NF-κB, whereas IL-10 and STAT3 were blocked. Rapamycin regulated the costimulatory/surface molecules CD86, programmed death ligand-1, and CD25 on mDCs and significantly increased the T cell allostimulatory potential of mDCs. In contrast, rapamycin suppressed immunostimulatory molecules and the allostimulatory potential of LPS-stimulated moDCs by an inability to augment NF-κB signaling. In differentiating moDCs, the PI3K/Akt-dependent mTOR pathway was constitutively activated by GM-CSF to induce DC differentiation in an mTORC1-dependent manner. Inhibition of mTORC1 or mTORC1/2 during moDC differentiation decreased moDC survival and markedly hampered its immunostimulatory phenotype. Analyzing the fate of DCs in vivo, we found that kidney transplant patients treated with rapamycin displayed an increased immunostimulatory potential of mDCs compared with patients treated with calcineurin inhibitors. Furthermore, rapamycin did not interfere with mDC differentiation in these patients. Collectively, mTOR exerts divergent immunoregulatory functions during DC activation and differentiation depending on the DC type that lead to opposing T cell responses, which might be of clinical importance in transplantation, cancer, and also for novel vaccination strategies.

Neovascular Age-related Macular Degeneration: Intraocular Cytokines and Growth Factors and the Influence of Therapy with Ranibizumab

PURPOSE To investigate concentrations of growth factors and inflammatory cytokines in eyes with neovascular age-related macular degeneration (AMD) before and during therapy with intravitreal ranibizumab and to identify associations with disease activity. DESIGN Prospective clinical trial. PARTICIPANTS AND CONTROLS Twenty-eight eyes of patients with neovascular AMD were compared with 28 eyes of age-matched patients with cataract as control. METHODS Ranibizumab was administered intravitreously once at baseline, and retreatments were given at monthly visits if optical coherence tomography (OCT) revealed macular edema or vision loss had occurred. Aqueous humor samples were taken each time intravitreal interventions were performed. Follow-up was 12 months. Luminex (Luminex Inc., Austin, TX) multiplex assays were used for measurement of 29 different growth factors and cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). MAIN OUTCOME MEASURES Differences in the concentrations of growth factors and inflammatory cytokines in eyes with neovascular AMD compared with control eyes and the influence of therapy with intravitreal ranibizumab. RESULTS A significantly increased expression of VEGF (P = 0.033) and a significantly decreased expression of PDGF (P = 0.038) were measured in the aqueous humor of eyes with neovascular AMD. Furthermore, a significant decrease of VEGF (P<0.001) was observed after intravitreal injection of ranibizumab along with significant changes in visual acuity and central retinal thickness (P = 0.039 and P<0.001). During follow-up with a flexible regimen, a correlation was identified between increased VEGF levels and persistent or recurrent macular edema. Changes in PDGF levels were strongly associated with alterations in VEGF concentration. CONCLUSIONS Vascular endothelial growth factor and PDGF-AA seemed to be associated with disease activity of neovascular AMD. Intravitreal anti-angiogenic treatment with ranibizumab resulted in significantly decreased intraocular VEGF expression below physiologic levels compared with controls. This effect was measurable as long as 4 weeks after each injection and was prolonged by consecutive retreatment. With recurrence after discontinuation of treatment, VEGF levels increased again.

B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction

B7‐H3 belongs to the B7 superfamily, a group of molecules that costimulate or down‐modulate T‐cell responses. Although it was shown that B7‐H3 could inhibit T‐cell responses, several studies – most of them performed in murine systems – found B7‐H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7‐H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T‐cell subsets. We show that B7‐H3 does not costimulate human T cells. In the presence of strong activating signals, B7‐H3 potently and consistently down‐modulated human T‐cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre‐activated T cells. Furthermore, we demonstrate that B7‐H3–T‐cell interaction is characterised by an early suppression of IL‐2 and that T‐cell inhibition can be reverted by exogenous IL‐2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT‐2) has been recently described as costimulatory receptor of murine B7‐H3 we have extensively analysed interaction of human B7‐H3 with TREML2/TLT‐2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7‐H3.

Serum Amyloid A in Uremic HDL Promotes Inflammation

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.