Arne von Bonin

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

Dipeptidyl‐peptidase IV/CD26 on T cells: analysis of an alternative T‐cell activation pathway

Summary: CD26 is a proteolytic enzyme (dipeptidyl‐peptidase IV) with a wide tissue distribution and a unique specificity that was already described 27 years ago. CD26 is expressed on a fraction of resting T cells at low density but is strongly upregulated following T‐cell activation. Recent results indicate that CD 2 6 is a muitifunctional molecule that may have important functions on T cells and in the immune system. It is associated with molecules of immunological importance such as the protein tyrosine phosphatase CD45 and adenosine deaminase (ADA) on the cell surface. Synthetic inhibitors of the enzymatic activity of CD26 have been shown to suppress certain immune reactions in vitro and in vivo. An interesting feature of CD26 is its ability to transmit a transmembrane signal to trigger functional programs in T cells. This triggering requires crosslinking of CD26 on a cell membrane. The enzymatic activity of CD2 6 is not obligatory for the activation of T cells via CD26. Since CD26 is a type II membrane protein with only six intracellular amino acids, it must deliver its signal via a signal‐transducing molecule. Signaling is dependent on the expression of the T‐cell receptor (TCR) complex with a special need for a functional ζ‐chain. In this context the ζ‐chain of the TCR complex is required for CD26‐mediated signaling but, in contrast to other co‐stimulatory molecules such as the CD2 molecule, is not sufficient for triggering the T cell.

ISOLATION OF PROCESSED, H-2KB-BINDING OVALBUMIN-DERIVED PEPTIDES ASSOCIATED WITH THE STRESS PROTEINS HSP70 AND GP96

Stress‐induced proteins or heat shock proteins (HSP) of 96 kDa mass (gp96) and 70 kDa mass (HSP70) have been shown previously to elicit specific immunity to tumors from which they are isolated. This immunity is dependent on CD8+ cytotoxic T cells which are readily primed in vivo by immunization with HSP. The immunization capacity of HSP relies on their ability to bind antigenic peptides. Here we show that HSP70 and gp96 preparations purified from the ovalbumin (OVA)‐transfected cell line E.G7 are associated with processed H‐2Kb ‐binding peptides which contain the major H‐2Kb ‐associated epitope SIINFEKL (OVA257 –264). Our data show for the first time in the well‐defined OVA antigen system that not only endoplasmic reticulum‐resident HSP, like gp96, are associated with processed antigenic peptides but that also the cytosolic HSP70 protein forms complexes with major finally processed MHC‐binding epitopes.

NK Cells Stimulate Proliferation of T and NK Cells through 2B4/CD48 Interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.

Molecular analysis of CD26-mediated signal transduction in T cells.

CD26 or dipeptidylpeptidase IV (DPP IV) is a cell surface protease involved in T cell activation. It is a type II transmembrane glycoprotein consisting of a large extracellular part, a single transmembrane region and a short cytoplasmic tail without any common signalling motifs. To eluciate the mechanisms involved in CD26-mediated signalling we have constructed C-terminal deletion mutants of the human CD26 molecule and transfected them into murine T cell hybridomas. Stimulation experiments show that most of the extracellular part of CD26 can be deleted without affecting its costimulatory activity. The membrane proximal glycosylation rich region of CD26 is sufficient to transduce costimulatory signals. Activation of T cells via CD26, however, is not mediated by the important T cell receptor associated adaptor proteins LAT and TRIM as shown in colocalization assays.

Heat Shock Protein 60 Is Released in Immune-Mediated Glomerulonephritis and Aggravates Disease: In Vivo Evidence for an Immunologic Danger Signal

Heat shock proteins (Hsp) are ubiquitous intracellular proteins that can be released in various forms of cellular stress. Some Hsp, such as Hsp60, have been shown to stimulate directly T cell-mediated immune responses in vitro. Here, it is demonstrated that Hsp60 is released from the kidneys and excreted into the urine of mice with nephrotoxic nephritis (NTN), a model of rapidly progressive glomerulonephritis. For examining the functional relevance of Hsp60 release, this protein was injected into mice with subnephritogenic NTN, in which only transient proteinuria and minimal organ damage occur that do not progress to terminal kidney failure. Injection of Hsp60 strikingly aggravated disease, as evidenced by global glomerular necrosis, tubulointerstitial damage, and complete anuria after 10 to 12 d. This effect was mediated neither by endotoxin contaminations of Hsp60 nor by autologous antibodies. It was strictly T cell dependent but not associated with a systemic Th1/Th2 shift. Thus, Hsp60 is an endogenous mediator stimulating immune effector mechanisms that contribute to the progression of NTN. These findings demonstrate in vivo that Hsp60 fulfills criteria of immunologic danger signals and suggest that such signals may be involved in immune-mediated kidney disease.

Expression of CD83 in the murine immune system

CD83 is used as a marker for mature dendritic cells (DC) in man. We have developed a new monoclonal antibody (mAb), Michel-17, that specifically recognizes mouse CD83. We show that murine CD83 is expressed mainly on mature DC and on activated T cells. Histological analysis of serial spleen sections revealed a CD83 expression pattern resembling that of MIDC-8, a known murine DC marker molecule. In contrast to other costimulatory receptors, cross-linking of CD83 with the mAb Michel-17 on DC or T cells does not induce any activation signals. Our data describe for the first time the expression pattern of murine CD83, which is comparable to that of human CD83.The unique mAb Michel-17 will help to elucidate the biological functions of the CD83 molecule in more detail.

Activation of cytotoxic T cells in vitro by recombinant gp96 fusion proteins irrespective of the ‘fused’ antigenic peptide sequence

Heat shock proteins (HSP) like Hsp70 and gp96 are potent molecules to induce MHC class I-restricted cytotoxic T cells against antigens present in the cells from which the HSP were isolated. Fusion proteins consisting of mycobacterial Hsp70 covalently linked to antigenic peptide sequences are also capable of generating CTL specific for the peptide-encoded antigens. For effective CTL induction direct binding of the peptide or covalent association of the peptide in the case of antigenic fusion proteins is required. Since mycobacterial Hsp70 and eukaryotic Hsp70 differ significantly in their primary structure, and since gp96 compared to Hsp70 is more efficient in priming antigen specific CTL in our hands, we created fusion proteins consisting of His-tagged eukaryotic gp96 fused C-terminally to various peptide antigens. Here, we used antigenic sequences derived from the established ovalbumin (OVA) and beta-galactosidase (beta-GAL) model systems. We show that in vitro established OVA and beta-GAL specific CTL clones release TNF-alpha and IFN-gamma when incubated with recombinant gp96 irrespective of the antigenic peptide sequences hooked to the C-terminus of gp96. In contrast to gp96 preparations purified from beta-GAL expressing cell lines, recombinant gp96/beta-GAL fusion proteins were not able to generate beta-GAL-specific T cells in vivo. Possible explanations for the lack of antigen-specific immunogenicity of gp96 fusion proteins in vivo are discussed.

Partial agonism and independent modulation of T cell receptor and CD8 in hapten‐specific cytotoxic T cells

We recently demonstrated antagonism for hapten‐reactive T cells by altered hapten ligands. Here we investigated partial peptide‐ or hapten‐agonism and effects of antigen stimulation on the expression of TCR and the CD8 coreceptor using a set of DNP‐ or TNP‐peptide‐induced, H‐2Kb ‐restricted mouse CTL clones. Various Kb ‐binding TNP‐ and DNP‐peptides acted as partial agonists, cross‐reactively stimulating individual clones for cytotoxicity and IFN‐γ secretion, but failing to induce proliferation or TNF‐α production. Full agonism, i.e. activation of all possible functions, was usually restricted to those hapten‐peptide combinations used for the induction of the respective clones. Our data imply distinctive kinetic optima for TCR antigen contacts in the induction of the various T cell effector functions. Down‐regulation of TCR was efficiently induced by full, but with one exception not by partial, agonists, indicating the independence of cytotoxicity or IFN‐γ secretion from TCR modulation. On the other hand, a reduction of TCR expression induced by full agonists was usually not accompanied by synchronous down‐modulation of CD8 as reported by others for human T cells. In fact, three of four full agonists and all partial agonists markedly enhanced rather than reduced the expression of CD8. Increased CD8 surface levels enhanced cytolytic potential and increased cross‐reactivity patterns of individual clones. Brefeldin A blocked this CD8 induction by partial agonists, and in the case of full agonists resulted in a parallel reduction of both, TCR and CD8. Thus, antigenic stimulation of mouse T cells initially down‐modulates CD8 together with TCR, but the loss of coreceptor is over‐compensated by a signal for increased CD8 export.

Efomycine M: an inhibitor of selectins?

To the editor: In a recent issue of Nature Medicine, Schön et al. characterized efomycine M (Efo-M) isolated from Streptomyces BS1261 as a specific inhibitor of selectins1. The authors showed that Efo-M at low micromolar concentrations is able to block Eand P-selectin interactions with sialyl Lewisx (sLex)-bearing ligands, for example, carcinoembryonic antigen (CEA). Moreover, Efo-M inhibited selectin-mediated leukocyte rolling in vivo and substantially improved the clinical symptoms of two in vivo inflammatory skin models using transplanted human skin biopsies. Thus, Efo-M was reported to function as a panselectin antagonist with promising therapeutic potential. Following the hypothesis by Schön et al., we performed extended in vitro and in vivo investigations with Efo-M. Although in principle we could confirm the anti-inflammatory profile of Efo-M, our findings suggest a mode of action that is separate from pan-selectin inhibition. As shown in Figure 1a, highly purified Efo-M (Supplementary Fig. 1 online) does not compete with the binding of 3H-labeled, polyvalent sLex (3H–p-sLex) to E-, Land P-selectins, even at concentrations of 100 μM in scintillation proximity assays (SPAs)2. In contrast, an excess of unlabeled p-sLex as well as fucoidan (Supplementary Fig. 2 online) clearly inhibited the binding of 3H–p-sLex, whereas monovalent sLex competed with 3H–p-Lex only in binding to E-selectin (Supplementary Fig. 3 online), as described previously3. As the experimental setup of SPAs described in Figure 1a allows the quantification of binding only to the glycan binding site of selectins, we repeated the experiment with 3H-labeled Efo-M (Fig. 1b), which should allow the detection of Efo-M binding to a glycan-independent binding site. However, no specific (for example, Ca2+-dependent and selectin-specific) binding of 3H-labeled Efo-M to Land P-selectin could be detected. Moreover, measuring direct interactions of Efo-M to selectins in ELISA (Fig. 1c) as well as in highly sensitive Biacore experiments revealed only a weak binding of Efo-M to L-selectin at higher concentrations, and no specific binding of Efo-M to Eand P-selectin in this type of assay was detectable (Fig. 1d). In their original study and a subsequent publication4, Schön et al. showed a model-based alignment of Efo-M and sLex structures, suggesting a comparable orientation of hydroxyl groups in Efo-M and sLex, which are crucial for the adhesive function of selectin ligands. Our analysis using crystallized Efo-M and a liquidstate conformation analysis of Efo-M by nuclear magnetic resonance (NMR) spectroscopy provided no evidence that sLex and Efo-M share a three-point pharmacophore when binding to the selectin surface (Supplementary Fig. 4 online). These results as well as the lack of binding of Efo-M to selectins under different experimental conditions suggest an alternative explanation for the findings of Schön et al. Although we observed anti-inflammatory activity in our in vivo models as well (Fig. 1e), Efo-M clearly exhibited selectinindependent effects on immune cells at low micromolar concentrations. Ex vivo–stimulated spleen cells from dinitrofluorbenzene-challenged and Efo-M–treated mice produced less tumor necrosis factor (TNF)-α (Fig. 1f). Moreover, in the presence of Efo-M (10 μM), activated mouse spleen cells (data not shown) and human peripheral blood mononuclear cells (PBMCs) showed a 50% reduction in cell viability (Fig. 1g), and lipopolysaccharide (LPS) induced secretion of TNF-α (Supplementary Fig. 5 online). At concentrations of >10 μM, Efo-M induced apoptosis in more than 30% of purified human B and T cells (Fig. 1h) and inhibited the maturation of dendritic cells (Supplementary Fig. 6 online). Efo-M was cytotoxic in PBMC cultures at low micromolar concentrations (50% inhibitory concentration (IC50), 4 μM), indicating again the substantial inhibition of cell viability caused by Efo-M, whereas we observed induction of apoptosis in human endothelial cells with higher concentrations of Efo-M (IC50, 27 μM; Supplementary Fig. 7 online) and no apoptosis in human neutrophils (data not shown). Together, our findings suggest that a selectinantagonizing function is probably not responsible for the observed in vivo results by Schön et al. and raise questions regarding how Efo-M inhibits inflammatory processes in the skin.