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.
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.
- Andreas H. Guse
Calcium Signaling Group, Institute of Biochemistry and Molecular
Biology I: Cellular Signal Transduction, University Medical Centre
- Friedrich Koch-Nolte, Friedrich Haag
Institute for Immunology, University Medical
Centre Hamburg-Eppendorf, Germany
- Simon Fillatreau, Kai Hoehlig, Vassiliki Lampropoulou
Deutsches Rheumaforschungszentrum, Berlin, Germany
- DFG (German National Research Association)
- Minka Breloer
- Katja Lüthje
- Birte Kretschmer
- Svenja Ehrlich
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
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.
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.
- Christian Kurts, University Clinic
of Bonn, Germany
- Mildred Scheel Stifung
- Fritz Bender Stifung
- Minka Breloer
- Anke Osterloh
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
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
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
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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