Scientific staff
Dr. Anke Osterloh (-453)

PhD/Diploma students
Katja Lüthje (-453)
Birte Kretschmer (-453)


Technical staff
Svenja Ehrlich (-453)

1) CD83: a regulator of lymphocyte maturation, function and homeostasis

Summary
The murine transmembrane glycoprotein is known to be an important regulator for both: thymic T cell maturation and peripheral T cell responses. Here, we analyze the role of CD83 in the regulation of B cells. While CD83 is not present on pro- and pre-B cells, immature and mature naïve B cells express low levels of CD83 that is rapidly upregulated upon activation in vivo and in vitro. Transgenic (tg) overexpression of CD83 in vivo leads to dramatically reduced and delayed Immunoglobulin (Ig) responses to T cell dependent (TD) and T cell independent (TI) model antigens and to infectious agents.
The defect is restricted to the B cell population since the antigen-specific T cell response of CD83tg mice to Leishmania major infection is unchanged. The defective Ig response is due to CD83 overexpression on the B cells themselves since CD83tg B cells do not respond to model antigen immunization in a mixed wild-type/CD83tg bone marrow chimera while wild-type B cells readily produce Ig within the very same mouse. CD83 tg B cells are further characterized by increased MHC-II and CD86 expression, reduced Ig secretion and increased IL-10 secretion in vitro while B cells with a severe reduction in CD83 expression (CD83mu) display the reciprocal phenotype.

CD83 overexpression on B cell precursors results in an impaired maturation of follicular B cells and a reduced survival of B cells in the periphery while the absence of CD83 increases peripheral survival of B cells. Finally the engagement of "naturally expressed" CD83 in wild type mice by injection with anti-CD83 mAb results in a dramatically increased IgG1 response to TI model antigen immunization.
Taken together our data strongly suggest that activation-induced CD83 on B cells confers negative signals as part of a regulatory feedback loop to prevent overstimulation of the B cell population as depicted in figure 1.




Figure 1: Hypothesis: CD83 as activation-induced negative regulator on B cells
A (1) Resting wild type B cells express only background levels of CD83. (2) Upregulation of CD83 upon activation by TLR ligands or B cell receptor signals in vivo and in vitro. (3) CD83 expression may render B cells susceptible for the reception of negative signals (tonic or ligand-induced signaling). Negative signals contribute to regulatory mechanisms preventing overstimulation of the B cell population.
B (1) CD83tg B cells overexpress CD83 constitutively, MHC-II and CD86 expression is increased. (2) CD83 overexpression may lead to the increased reception of negative signals. As a consequence Ig secretion in vivo and in vitro is reduced, IL-10 secretion is increased, B cell survival in vivo is decreased.
C: (1) Resting CD83 mutant (CD83mu) B cells display no CD83 expression, MHC-II and CD86 expression is reduced. (2) CD83 mutant B cells show strongly reduced CD83 upregulation upon activation and are thus less susceptible for the reception of negative signals. Therefore they display slightly increased Ig and reduced IL-10 secretion. B cell survival in vivo is increased.

Cooperating partners

• Andreas H. Guse
  Calcium Signaling Group, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, University Medical Centre Hamburg-Eppendorf, Germany
• Friedrich Koch-Nolte, Friedrich Haag
  Institute for Immunology, University Medical
  Centre Hamburg-Eppendorf, Germany
• Simon Fillatreau, Kai Hoehlig, Vassiliki Lampropoulou
  Deutsches Rheumaforschungszentrum, Berlin, Germany

Funding

• DFG (German National Research Association)

Investigators

• Minka Breloer
  
• Katja Lüthje
  
• Birte Kretschmer
  
• Svenja Ehrlich
  

Publications

Lüthje, K, Kretschmer B., Fleischer B. and M. Breloer 2008.
CD83 regulates splenic B cell maturation and peripheral B cell homeostasis
International Immunol in press

Breloer M and B. Fleischer 2008. CD83 regulates lymphocyte maturation, activation and homeostasis. Trends Immunol. 29: 186

Breloer M 2008. CD83: regulator of central T cell maturation and peripheral immune response. (Invited Letter to the Editor) Immunol Lett. 115: 9

Reinwald, S., C. Wiethe, A. M. Westendorf, M. Breloer, M. Probst-Kepper, B. Fleischer, A. Steinkasserer, J. Buer, and W. Hansen. 2008. CD83 expression in CD4+ T cells modulates inflammation and autoimmunity. J Immunol 180:5890-5897.

Breloer, M., B. Kretschmer, K. Lüthje, S. Ehrlich, U. Ritter, T. Bickert, C. Steeg, S. Fillatreau, K. Hoehlig, V. Lampropoulou, and B. Fleischer. 2007. CD83 is a regulator of murine B cell function in vivo. Eur J Immunol. 37: 634

Kretschmer, B., Lüthje K., Guse A. H., Ehrlich S., Koch-Nolte F., Haag F., and M. Breloer. 2007. CD83 modulates B cell function in vitro: increased IL-10 and reduced Ig secretion by CD83tg B cells. PloS ONE 2 (8): e755.

Lüthje, K., Cramer, S.O., Ehrlich S., Veit, A., Steeg, C. Fleischer, B., von Bonin A. and M. Breloer. 2006. Transgenic expression of a CD83 immunoglobulin fusion protein impairs the development of immune competent CD4 positive T cells. Eur J Immunol 36: 2035

2) Autologous Heat Shock Protein 60: A danger signal to the immune system

Summary
A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past. Hsp60 has been shown to induce the secretion of pro-inflammatory cytokines in professional antigen presenting cells via TLR-4 signalling and to enhance the activation of T cells in primary stimulation. However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations. Employing LPS-depleted Hsp60 and Hsp60 expressed on the surface of eukaryotic cell lines, we were able to dissect the Hsp60 protein mediated effects from biologic effects that are mediated by prokaryotic contaminants for the first time. We show that LPS-free Hsp60 still induces T cell activation, but in contrast to LPS-contaminated Hsp60 this activation is not mediated by TNF-a, IL-12 or IL-6 induction and does not depend on TLR-4 signalling. The immunstimmulatory effect of Hsp60 is relevant in vivo since mice transplanted with Hsp60-positive tumors show a prolonged survival compared to mice transplanted with wild type tumors. Taken together we provide evidence that autologous Hsp60 has a biological function that is not due to contaminating pathogen-associated molecules. Therefore, we propose two different biological mechanisms by which Hsp60 modulates immune responses, one in association with LPS in bacterial infection and another in the absence of LPS in the situation of virus induced or stress induced non-apoptotic cell death.

Project Results
1. LPS-free Hsp60 enhances IFN-g production in the primary stimulation of T cells independent of IL-12 and TLR-4 signalling
We employed LPS-depleted recombinant Hsp60 and Hsp60 expressed on the cell surface of the murine cell line X63 to analyze the stimulatory capacity of Hsp60 in the absence of LPS. Addition of LPS-free Hsp60 to a T-cell stimulation culture did not induce transcription or translation of the cytokines IL-12 p70, p35, p40, TNF-a and IL-6 in the APC but still led to a significant increase of IFN-g production in T cells (data not shown and figure 1A). By using T cells and APC derived from the TLR-4 mutant mouse strain C3H/HeJ we analyzed the role of functional TLR-4 signalling in Hsp60 mediated costimulation. Figure 1A shows that Hsp60 still increased the IFN-g production in a T cell stimulation culture even if either the T cell, or the APC or both cell types carried a dysfunctional TLR-4. LPS mediated T cell activation in contrast was strictly dependent on the expression of a functional TLR-4 receptor (figure 1B).

Fig 1: Hsp60 mediated T cell activation is independent of TLR-4
5x104 T cells and 1x105 macrophages prepared from TLR-4 mutant C3HheJ and syngenic C3HheN mice were cultured in indicated combinations in the presence of anti CD3 mAb (145 2C11 0.3µg/ml). Either 1x104 cells of two different Hsp60 expressing X63 clones (black bar, grey bar 1A) or mock transfected X63 cells (white bar 1A) or LPS (1µg/ml, black bar 1B) or medium (white bar 1B) was added. IFN-g content in the supernatant was analyzed after 24h culture. (* p<0.05, ** p<0.005, *** p<0.0005)

2. More than one mechanism for the Hsp60 mediated T cell activation
Hsp60 has been shown to bind LPS and to enhance LPS mediated cytokine production in macrophages. Therefore, in bacterial infection Hsp60 released by dying cells in vivo may function as an LPS carrier protein that facilitates binding of LPS to the CD14/TLR-4 receptor complex and enhances LPS mediated TLR-4 signalling, similar to the LPS binding protein LBP. This situation would resemble the experiments employing recombinant Hsp60 contaminated with endotoxin in vitro. In the absence of LPS, for example in a viral infection or in situations of necrotic cell death Hsp60 itself may act immunstimulatory, employing a TLR-4 independent signalling pathway. This pathway does not lead to the secretion of cytokines like IL-12 by macrophages but, nevertheless, stimulates the production of IFN-g in T cells.

Fig. 2: Different mechanisms of Hsp60 mediated T cell activation
Hypothetical mechanism of T cell activation by Hsp60 in the presence (left panel) or in the absence (right panel) of LPS.

3) A role for Hsp60 in tumor immunology?

In order to investigate the impact of Hsp60 on tumor rejection we compared the immune response to Hsp60-negative and Hsp60-positive X63 cells. Immunization with different Hsp60 transfected X63 cells led to a significant higher frequency of IFN-g secreting spleen cells (data not shown). Mice that were transplanted with Hsp60-positive tumor cells showed prolonged survival (figure 3). Taken together our data suggest that over expression of autologous Hsp60 renders tumors more immunogenic in vivo.

Fig. 3: Hsp60-positive tumors are more immunogenic than Hsp60 negative tumors
1´104 Hsp60-negative (X63-pFM92) or -positive X63 cells (X63-mHsp60#12, X63-mHsp60#17) were injected intravenioulsy in BALB/c-mice. Survival was monitored and is indicated as % survial on the y-axis.

Cooperating partners

• Christian Kurts, University Clinic of Bonn, Germany

Funding

• Mildred Scheel Stifung
• Fritz Bender Stifung

Investigators

• Minka Breloer
• Anke Osterloh
  

Publications

Osterloh A., Veit A., Gessner A., Fleischer B., Breloer M 2008. Hsp60-mediated T cell stimulation is independent of TLR4 and IL-12. Int Immunol. 20: 433

Osterloh, A. and M. Breloer. 2008. Heat shock proteins: linking danger and pathogen recognition. Med Microbiol Immunol. 197: 1

Lang, A., D. Benke, F. Eitner, D. Engel, S. Ehrlich, M. Breloer, E. Hamilton-Williams, S. Specht, A. Hoerauf, J. Floege, A. von Bonin, and C. Kurts. 2005. Heat shock protein 60 is released in immune-mediated glomerulonephritis and aggravates disease: in vivo evidence for an immunologic danger signal. J Am Soc Nephrol 16:383.

Osterloh, A., F. Meier-Stiegen, A. Veit, B. Fleischer, A. Von Bonin, and M. Breloer. 2004. LPS-free heat shock protein 60 activates T cells. J Biol Chem. 279: 47906

Arne von Bonin, Minka Breloer and Solveig More. 2003. Eucarytotic Hsp60: A danger signal for T- and natural killer cells. In Heat shock proteins and inflammation. W. v. Eden, ed. Birkhäuser Verlag, Basel, p. 55.

von Bonin, A., S. H. More, and M. Breloer. 2003. Purification of the eucaryotic heat-shock proteins Hsp70 and gp96. Methods Mol Biol 215:193.

Breloer, M., S. H. More, A. Osterloh, F. Stelter, R. S. Jack, and A. Bonin Av. 2002. Macrophages as main inducers of IFN-gamma in T cells following administration of human and mouse heat shock protein 60. Int Immunol 14:1247.

More, S. H., M. Breloer, A. K. Fentz, B. Fleischer, and A. von Bonin. 2002. 'Ignorance' of antigen-specific murine CD4+ and CD8+ T cells is overruled by lipopolysaccharide and leads to specific induction of IFN-gamma. Scand J Immunol 55:329.

Breloer, M., B. Dorner, S. H. More, T. Roderian, B. Fleischer, and A. von Bonin. 2001. Heat shock proteins as "danger signals": eukaryotic Hsp60 enhances and accelerates antigen-specific IFN-gamma production in T cells. Eur J Immunol 31:2051.

More, S. H., M. Breloer, and A. von Bonin. 2001. Eukaryotic heat shock proteins as molecular links in innate and adaptive immune responses: Hsp60-mediated activation of cytotoxic T cells. Int Immunol 13:1121.

Breloer, M., B. Fleischer, and A. von Bonin. 1999. In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins. J Immunol 162:3141.

More, S., M. Breloer, B. Fleischer, and A. von Bonin. 1999. Activation of cytotoxic T cells in vitro by recombinant gp96 fusion proteins irrespective of the 'fused' antigenic peptide sequence. Immunol Lett 69:275.

Breloer, M., T. Marti, B. Fleischer, and A. von Bonin. 1998. Isolation of processed, H-2Kb-binding ovalbumin-derived peptides associated with the stress proteins HSP70 and gp96. Eur J Immunol 28:1016.

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