CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock.
ABSTRACT CD6 is a lymphocyte receptor that belongs to the scavenger receptor cysteine-rich superfamily. Because some members of the scavenger receptor cysteine-rich superfamily act as pattern recognition receptors for microbial components, we studied whether CD6 shares this function. We produced a recombinant form of the ectodomain of CD6 (rsCD6), which was indistinguishable (in apparent molecular mass, antibody reactivity, and cell binding properties) from a circulating form of CD6 affinity-purified from human serum. rsCD6 bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of lipoteichoic acid and LPS, respectively. The Kd of the LPS-rsCD6 interaction was 2.69 +/- 0.32 x 10(-8) M, which is similar to that reported for the LPS-CD14 interaction. Further experiments showed that membrane CD6 also retains the LPS-binding ability, and it results in activation of the MAPK signaling cascade. In vivo experiments demonstrated that i.p. administration of rsCD6 before lethal LPS challenge significantly improved mice survival, and this was concomitant with reduced serum levels of the proinflammatory cytokines TNF-alpha, IL6, and IL-1beta. In conclusion, our results illustrate the unprecedented bacterial binding properties of rsCD6 and support its therapeutic potential for the intervention of septic shock syndrome or other inflammatory diseases of infectious origin.
- SourceAvailable from: jimmunol.org[show abstract] [hide abstract]
ABSTRACT: Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.The Journal of Immunology 02/2002; 168(2):554-61. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Antigen presenting cells (macrophages and dendritic cells) express pattern recognition molecules that are thought to recognize foreign ligands during early phases of the immune response. The best known of these are probably the Toll-like receptors, but a number of other receptors are also involved. Several of these recognize endogenous as well as exogenous ligands, suggesting that they play a dual role in normal tissue function and host defense.Cell 01/2003; 111(7):927-30. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier. X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype). At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces. As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained. At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory. At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype. The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype. In excess buffer, the addition of divalent cations brought the bilayers into steric contact. Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core. The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids. This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.Biochemistry 09/1999; 38(33):10758-67. · 3.38 Impact Factor
CD6 binds to pathogen-associated molecular patterns
and protects from LPS-induced septic shock
Maria-Rosa Sarrias*, Montserrat Farno ´s*, Rube ´n Mota†, Fernando Sa ´nchez-Barbero‡, Anna Iba ´n ˜ez*, Idoia Gimferrer*,
Jorge Vera*, Rafael Fenutrı ´a*, Cristina Casals‡, Jose ´ Ye ´lamos§, and Francisco Lozano*¶
*Servei d’Immunologia, Hospital Clı ´nic de Barcelona, Institut d’Investigacions Biome `diques August Pi i Sunyer, Facultat de Medicina, Universitat de
Barcelona, 08036 Barcelona, Spain;‡Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid,
28040 Madrid, Spain;†Department of Biochemistry, Molecular Biology, and Immunology, School of Medicine, University of Murcia, 30120 Murcia,
Spain; and§Department of Immunology, Municipal Institute of Medical Research, Barcelona Biomedical Research Park, 08003 Barcelona, Spain
Edited by Philippa Marrack, National Jewish Medical and Research Center, Denver, CO, and approved May 29, 2007 (received for review March 26, 2007)
CD6 is a lymphocyte receptor that belongs to the scavenger
receptor cysteine-rich superfamily. Because some members of the
scavenger receptor cysteine-rich superfamily act as pattern recog-
nition receptors for microbial components, we studied whether
CD6 shares this function. We produced a recombinant form of the
ectodomain of CD6 (rsCD6), which was indistinguishable (in ap-
parent molecular mass, antibody reactivity, and cell binding prop-
erties) from a circulating form of CD6 affinity-purified from human
serum. rsCD6 bound to and aggregated several Gram-positive and
-negative bacterial strains through the recognition of lipoteichoic
acid and LPS, respectively. The Kdof the LPS–rsCD6 interaction was
2.69 ? 0.32 ? 10?8M, which is similar to that reported for the
LPS–CD14 interaction. Further experiments showed that mem-
brane CD6 also retains the LPS-binding ability, and it results in
activation of the MAPK signaling cascade. In vivo experiments
demonstrated that i.p. administration of rsCD6 before lethal LPS
challenge significantly improved mice survival, and this was con-
comitant with reduced serum levels of the proinflammatory cyto-
kines TNF-?, IL6, and IL-1?. In conclusion, our results illustrate the
unprecedented bacterial binding properties of rsCD6 and support
its therapeutic potential for the intervention of septic shock syn-
drome or other inflammatory diseases of infectious origin.
bacterial cell component ? innate immunity ? lymphocyte surface receptor
acterized by the presence of one or several repeats of a cysteine-
rich extracellular domain named SRCR (1, 2). The SRCR-SF
includes both cell-surface and secreted proteins that can be
expressed on cells of hemopoietic origin such as macrophages
(e.g., SR-AI/II, MARCO, CD163, Mac2-binding protein, and
Sp?) and lymphocytes (e.g., CD5, CD6, and T19/WT1), as well
as on nonhematological cells such as those from of the digestive,
respiratory, and urinary epithelial tracts (e.g., DMBT1, S4D-
SRCRB, and SCARA5) (2). There is no unifying function for all
of the members of the SRCR-SF, but some of them have been
implicated in the development of the immune system and in the
regulation of innate and adaptive immune responses (3). A few
members of the SRCR-SF (i.e., SR-AI/II, MARCO, DMBT1,
Sp?, and SCARA5) are known to interact with bacteria and to
bind to conserved pathogen-associated molecular patterns
present on microbial surfaces, such as LPS, lipoteichoic acid
(LTA), and peptidoglycan. These interactions were initially
mapped outside the SRCR domains (4), but recent reports have
demonstrated the direct involvement of the SRCR domains in
such interaction (5–8). Given the conserved structure of SRCR
domains throughout the evolution, it remains to be analyzed
whether pathogen scavenging is a general property shared by all
members of the SRCR-SF or only by a selected group of its
The CD5 and CD6 receptors are the two only members of the
SRCR-SF that are expressed on human lymphocytes. Both are
he scavenger receptor cysteine-rich superfamily (SRCR-SF)
is an ancient and highly conserved family of proteins, char-
lymphoid-specific surface glycoproteins sharing important sim-
CD6 are expressed on thymocytes, mature T cells, and the B1a
B cell subset, although CD6 expression has also been reported
on certain regions of the brain (9). They exhibit important
differences in their cytoplasmic regions, but their extracellular
regions are exclusively composed of three consecutive SRCR
domains, which show extensive amino acid sequence identity (1,
10). Functionally, they are physically associated to the antigen-
specific receptor complex present on T (TCR/CD3) and B
(BCR) cells, where they contribute to either positive or negative
by that receptor complex (2, 11, 12). In the present study we have
explored the bacterial binding capabilities of the ectodomains of
the human lymphocyte receptors CD5 and CD6, both known to
exist as membrane receptors, but also as soluble receptors
circulating in serum (13, 14). Data are provided herein on the
binding of soluble and membrane forms of CD6, but not CD5,
to the surface of Gram-positive and Gram-negative bacteria
through the recognition of pathogen-associated molecular pat-
terns (namely, LPS and LTA). The relevance of such an inter-
action is illustrated by the beneficial effects of the infusion of a
recombinant soluble form of CD6 (rsCD6) on the survival rate
in a mouse experimental model of septic shock.
rsCD6 Binds to Gram-Positive and Gram-Negative Bacteria. To de-
termine whether the ectodomain of human CD6 and CD5 could
directly bind to the surface of whole bacteria, we used an
to bacteria (15). Thus, biotin-labeled recombinant soluble pro-
teins encompassing the ectodomains of human CD5, CD6, and
Sp? (rsCD5, rsCD6, and rSp?) (Fig. 1A) were incubated with
bacterial suspensions, and their binding to bacterial pellets was
further assayed by SDS/PAGE and Western blotting against
streptavidin–HRP. Our results show that, as previously reported
for rSp? (8), rsCD6 bound to Gram-positive and -negative
bacteria (Fig. 1B), indicating that this protein also possesses
bacterial binding activity. In contrast, neither rsCD5 nor the
negative control BSA bound to bacterial suspensions. As illus-
Author contributions: M.-R.S. and M.F. contributed equally to this work; M.-R.S., C.C., J.Y.,
I.G., C.C., and J.Y. contributed new reagents/analytic tools; M.-R.S., R.M., F.S.-B., C.C., J.Y.,
and F.L. analyzed data; and M.-R.S., C.C., J.Y., and F.L. wrote the paper.
Conflict of interest statement: This work is the subject of patent application ES200700893
submitted by the University of Barcelona.
This article is a PNAS Direct Submission.
Abbreviations: SRCR-SF, scavenger receptor cysteine-rich superfamily; LTA, lipoteichoic
acid; TLR, Toll-like receptor.
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
July 10, 2007 ?
vol. 104 ?
trated by Fig. 2C, the presence of biotin-labeled rsCD6 was
greatly reduced in Escherichia coli and Staphylococcus aureus
bacterial cell pellets in the presence of EDTA. This indicates
that, like rSp? (8) and CRP-ductin (16), rsCD6 recognition of
cell wall components from Gram-positive and -negative bacteria
is facilitated by Ca2?.
We next sought to determine whether the observed binding of
rsCD6 to bacteria was specific and to identify which bacterial
cell-surface structures were being recognized. To answer these
questions, competition experiments were designed in which
biotin-labeled rsCD6 was incubated with increasing concentra-
tions of purified LPS or LTA before the addition of a suspension
of either E. coli or S. aureus (5 ? 107cells). LPS and LTA were
assayed because they are ubiquitous cell-surface components of
these microorganisms. As illustrated by Fig. 2D, binding of
biotin-labeled rsCD6 to E. coli was competed in a dose-
dependent manner by LPS (from E. coli), but not by LTA (from
S. aureus). On the contrary, when the binding of rsCD6 to S.
aureus was studied, LPS did not affect such interaction. Inter-
estingly, this binding was competed in a dose-dependent manner
by LTA from S. aureus.
Purification of nsCD6 from Human Serum. Purification yielded 6 ?g
SDS/PAGE analysis and Coomassie blue staining (Fig. 2A). The
observed molecular mass closely resembles that of recombinant
soluble CD6 (rsCD6) (17), which is exclusively composed of the
of the membrane form of CD6 (mCD6), which ranges from 105 to
130 kDa depending on its degree of phosphorylation (18). The
observed molecular mass of the three different CD6 forms, i.e.,
rsCD6, nsCD6, and mCD6, immunoprecipitated from HUT-78 T
cells is shown in Fig. 2B. The purified nsCD6 protein was identified
as CD6 by Western blotting assays with a polyclonal antiserum
raised against the extracellular region of human CD6 (Fig. 2C).
Interestingly, mCD6, but not rsCD6 or nsCD6, was reactive with a
polyclonal antiserum raised against the intracytoplasmic region of
human CD6 (12) (data not shown). In cell binding experiments,
to K562 erythroleukemic cells, in accordance with the differential
expression of the CD6 ligand (ALCAM/CD166) (17, 19) (Fig. 2D).
results presented in Fig. 3A show that, in accordance with the
bacterial binding experiments in Fig. 1, both natural and recombi-
nant soluble CD6 forms bound to plastic-coated LPS in a dose-
dependent fashion. No BSA–LPS interaction could be observed.
The binding of rsCD6 and rsCD5 to a rough mutant (Re595) of
in fluorescent properties of FITC–Re-LPS such as anisotropy and
intensity. Fig. 3B shows the binding of rsCD6 and rsCD5 to
LPS molecule. Fluorescence anisotropy measurements depend on
Western blot analysis of the affinity-purified biotin-labeled proteins. (B)
Protein binding to E. coli and S. aureus. (C) Calcium influence on the binding
of rsCD6 and rSp? to E. coli and S. aureus. (D) Competition binding assays of
rsCD6 to E. coli and S. aureus in the presence of increasing concentrations of
LPS or LTA. For bacterial binding studies, biotin-labeled proteins were incu-
buffer and electrophoresed. Detection of biotin-labeled proteins was per-
formed by Western blot using HRP–streptavidin.
Binding of rsCD6 to Gram-positive and Gram-negative bacteria. (A)
serum. (A) Coomassie blue staining of affinity-purified nsCD6 from human
serum. (B) Western blot analysis of biotin-labeled purified nsCD6 and rsCD6
proteins and membrane CD6 (mCD6) immunoprecipitated from surface bio-
tinylated HUT-78 cells with streptavidin–HRP. (C) Membranes containing the
same proteins as in B, Western blotted with a rabbit polyclonal antiserum
specific for the extracellular region of CD6. (D) Flow cytometry analysis of the
reactivity of biotinylated rsCD5, rsCD6, nsCD6, or BSA (used as a negative
control) with the K652 and Raji cells. Bound protein was detected with
Characterization of affinity-purified circulating CD6 from human
and rsCD6 to LPS. Several concentrations of biotinylated rsCD6, nsCD6, or BSA
(as negative control) were added to the LPS-coated wells, and bound protein
was detected with streptavidin–HRP. (B and C) Binding of rsCD6 or rsCD5 to
Re-LPS was monitored by changes in FITC–Re-LPS fluorescent properties. (B)
upon binding to FITC–Re-LPS, which increases with increasing rsCD6 concen-
nm upon addition of increasing amounts of rsCD6 or rsCD5. The apparent Kd
a rectangular hyperbola, was 2.69 ? 0.32 ? 10?8M.
Binding of rsCD6 to LPS. (A) ELISA showing direct binding of nsCD6
Sarrias et al.PNAS ?
July 10, 2007 ?
vol. 104 ?
no. 28 ?
the rate and extent of the rotational motion of the fluorophore
during the lifetime of the excited state. Addition of different
amounts of rsCD6 to FITC–Re-LPS caused a protein concentra-
tion-dependent increase of the anisotropy values of FITC–Re-LPS,
indicating that the binding of rsCD6 to Re-LPS caused mechanical
restrictions of the rotational mobility of the dye. Control experi-
ments were done with free fluorescein to demonstrate that all of
these changes did not result from the interaction of rsCD6 with
fluorescein, but with the LPS molecule; the fluorescence emission
anisotropy of free fluorescein was very low and was not affected by
addition of 3-fold excess of rsCD6 (data not shown). On the other
hand, rsCD5 did not cause any change in FITC–Re-LPS fluores-
cence anisotropy, indicating that this protein does not bind to
Addition of rsCD6, but not rsCD5, to FITC–Re-LPS in
intensity of fluorescent LPS. Fig. 3C shows that the magnitude
of the fluorescence intensity change at 520 nm increased as a
function of rsCD6 concentration, but not rsCD5 concentration,
and was saturable. These results allowed us to determine the
affinity of rsCD6 binding to LPS. The apparent Kdfor FITC–
Re-LPS/rsCD6 complexes, calculated from the saturation curve
fitted to a rectangular hyperbola, was 2.69 ? 0.32 ? 10?8M.
Binding of LPS to Cell-Surface CD6. We next questioned whether the
the cell surface. These studies were performed by staining with
FITC-labeled LPS of 2G5 cells, a Jurkat cell derivative selected for
deficient CD5 and CD6 expression (20). As shown in Fig. 4 A and
B, fluorescence intensity was higher in 2G5 cells stably expressing
wild-type CD6 (2G5-CD6.wt) (12) compared with parental un-
transfected 2G5 cells. Further confirmation of our results was
obtained from competition binding experiments. In these experi-
ments, binding of FITC–LPS to 2G5-CD6.wt cells was inhibited in
a dose-dependent manner by rsCD6, but not with rsCD5 or BSA
(data not shown), used as a negative control (Fig. 4C), indicating
that the inhibition was specific. 2G5 and 2G5-CD6.wt cells were
negative for CD14 expression but expressed equivalent amounts of
Toll-like receptor 4 (TLR4) on their surface (data not shown), in
binding cannot be attributed to differential expression of TLR4.
From these data we conclude that LPS is able to interact with CD6
on the cell surface.
Binding of LPS to Membrane CD6 Induces ERK1/2 Activation. Further
evidence of LPS binding to cell-surface CD6 was obtained from
activation of the MAPK signaling cascade in transient and stable
stimulation of 2G5-CD6.wt transfectants resulted in marked
ERK1/2 phosphorylation responses compared with parental 2G5
addition of PHA, the latter used as a positive control and to show
the integrity of the MAPK signaling cascade. Similar results were
obtained with transient transfection of CD6.wt in the heterologous
COS-7 cell system (Fig. 4E). Fig. 4E shows that COS-7 cells
transiently expressing a cytoplasmatic tail-truncated molecule
(CD6.P527stop) (22) failed to respond to LPS but not to PHA,
demonstrating that integrity of the cytoplasmic region of CD6 is
also required for proper LPS-induced ERK1/2 phosphorylation.
The differences in ERK1/2 phosphorylation are not due to differ-
ential CD6 surface expression, as assessed by FACS analysis and
Western blot experiments (data not shown).
Binding of rsCD6 Leads to both Bacteria and LPS Aggregation. We
hypothesized that the existence of multiple bacterial binding
domains on the rsCD6 molecule would lead to bacterial aggre-
gation phenomena. Fig. 5A shows that presence of rsCD6
induced aggregation of Gram-negative (E. coli) as well as
Gram-positive (S. aureus) bacteria, to a similar extent as the
positive control rSp? (8). In accordance with its inability to bind
bacteria, rsCD5 was also unable to induce their aggregation, and
this also was the case of the negative control HSA.
cytometry analysis showing direct binding of increasing amounts of LPS–FITC to
parental and CD6.wt-transfected 2G5 cells. (B) To ease comparison, mean fluo-
rescence intensities of A were plotted against the amount of LPS–FITC added to
each cell line. (C) Competition studies of LPS–FITC binding to the 2G5-CD6.wt
transfectants. Cells were incubated with 10 ?g of LPS–FITC in the presence of
increasing amounts of rsCD6 or rsCD5. (D and E) Analysis of ERK1/2 phosphory-
(D) and COS-7 cells transiently expressing wild-type (CD6.wt) or cytoplasmic
were stimulated for 40 min with 100 ?g/ml LPS or 100 ng/ml PHA at 37°C. Cell
immunoblotting with anti-phospho ERK1/2 (p-ERK1/2) antiserum. Further rep-
robing with anti-ERK1/2 antiserum was used as loading control.
LPS from E. coli binds to cell-surface CD6 and activates ERK1/2. (A) Flow
aureus bacterial suspensions were incubated overnight at room temperature
with rsCD6 or rsCD5 (2 ?M) in the presence of 5 mM Ca2?. Equimolar concen-
trations of rSp? and HSA were used as positive and negative control, respec-
tively. Aggregation was observed by direct examination on a fluorescence
were 100 ?g/ml, 2.5 mM, and 5 mM, respectively. One representative exper-
iment of two performed is shown.
rsCD6 induces bacterial aggregation. (A) FITC-labeled E. coli and S.
www.pnas.org?cgi?doi?10.1073?pnas.0702815104Sarrias et al.
We further explored the process of Re-LPS aggregation
induced by Ca2?in the presence of rsCD6. This was analyzed by
measuring changes in light absorbance at 400 nm (Fig. 5B).
These experiments were carried out under the same ionic
conditions as binding studies with fluorescent LPS, except that
Ca2?as well as concentrations of Re-LPS 200 times higher were
needed to produce detectable light absorption at 400 nm. Fig. 5B
shows that LPS molecules were able to aggregate in buffers
containing Ca2?and that low concentrations of rsCD6 induced
a further aggregation of LPS. Control assays showed that
addition of Ca2?(2.5 mM) does not affect the aggregation status
at 275 nm (data not shown). Together, these data suggest that,
in the presence of Ca2?, rsCD6 may contribute to increase the
size of bacterial aggregates as well as of LPS aggregates.
rsCD6 Prevents LPS-Induced Septic Shock in Mice. We assessed
whether administration of rsCD6 into mice would improve their
6A, administration of a single i.p. dose of 25 ?g of rsCD6, but not
rsCD5, in mice 1 h before i.p. LPS challenge significantly
enhanced their survival rate as compared with the saline control
(up to 70%). In accordance with these data, administration of
rsCD6 induced a significant reduction on the levels of plasma
proinflammatory cytokines TNF-?, IL-1?, and IL-6 in these
mice (Fig. 6 B, C, and D, respectively).
In the present study it is shown that human CD6, a cell-surface
receptor mainly expressed by cells of the lymphoid lineage, is
able to bind to conserved bacterial structures such as LPS and
LTA. Interestingly, the Kdof the LPS–CD6 interaction appeared
to be of relative high affinity, similar in magnitude to that
reported for the interaction of LPS with CD14, the most widely
accepted LPS receptor in mammalian cells. These data are of
relevance by themselves to the knowledge of the biological role
of the CD6 receptor, but also because they led to the finding that
i.p. administration of a recombinant form of CD6 abolished the
lethal effects of LPS-induced septic shock in mice.
Innate immune responses rely on the ability of multiple non-
polymorphic germ-line-encoded receptors to recognize the so-
called pathogen-associated molecular patterns, which are con-
served products of microbial pathogens not shared by the host and
essential for their survival (23). Pattern-recognition receptors are
mainly expressed by phagocytic cells (granulocytes, macrophages,
and dendritic cells) and cells of epithelial barriers (23). However,
recent studies report the expression of pattern-recognition recep-
tors, namely TLR, in cells central to adaptive immune responses
such as T and B lymphocytes (21, 24, 25). Among the receptors
involved in pattern recognition, there are several members of the
SRCR-SF expressed on macrophages and mucosal surfaces (5, 8,
16, 26). As far as we know, no information is currently available on
that regard for the two only members of the SRCR-SF expressed
we have shown that CD6, but not CD5, can bind to Gram-positive
and -negative bacteria (Fig. 1) and that this binding was facilitated
by the presence of Ca2?. CD6 is not a type C lectin, and it does not
aggregate in the presence of Ca2?. On the contrary, presence of
Ca2?is known to affect the structure of the bacterial external
may facilitate the rsCD6–LPS interaction through its effect on
bacteria and LPS. Competition binding experiments showed that
the observed interaction of rsCD6 with the bacterial surfaces is
specific. They also indicate that, like Sp? (8), rsCD6 binds to LTA
and LPS through independent and nonoverlapping sites of the
Low levels of soluble CD6 have been detected in normal
human sera (14), but its biochemical characterization has not
been achieved until present. We have purified from pooled
human sera a natural soluble CD6 (nsCD6) protein with mo-
lecular mass, antibody reactivity, and cell binding characteristics
similar to rsCD6 (17) (Fig. 2). These data, together with the
shared ability to bind to LPS in ELISAs (Fig. 3), suggest that
rsCD6 retains the biological activities of the circulating form of
CD6 (nsCD6). These results validate the use of rsCD6 in the
ensuing studies given the low availability of nsCD6.
The interaction of rsCD6 with LPS has an apparent Kd of
2.69 ? 0.32 ? 10?8M. Using the same methodology and the
same ionic conditions, Kdvalues for FITC–Re-LPS/LBP, FITC–
Re-LPS/sCD14, FITC–Re-LPS/SP-A, and FITC–Re-LPS/rSp?
complexes are 3.5 ? 10?9M, 2.9 ? 10?8M, 2.8 ? 10?8M, and
1.83 ? 10?7M, respectively (8, 28, 29). These data indicate that
rsCD6 binds to Re-LPS ?10-fold more tightly than SRCR-
containing rSp? but with affinity similar to CD14 (28).
In contrast to rsCD6, the recombinant form encompassing the
ectodomain of CD5 (rsCD5) did not bind to bacteria (Fig. 1) or
LPS (Fig. 3). It seems, therefore, that the SRCR domains of CD6
still retain the bacterial binding properties shared by some
members of the SRCR-SF, whereas those of CD5 do not. This
suggests that CD5 and CD6 may have followed a divergent
evolution, which is of relevance for the function of these
receptors and the SRCR structure.
Although CD6 is found circulating in serum, this receptor is
mainly found expressed on the cell surface. Enhanced binding of
LPS–FITC to the surface of 2G5 cell transfectants expressing
CD6 versus the parental cells suggested that membrane-bound
CD6 also retains the ability to bind to LPS (Fig. 4 A and B).
Moreover, the ability of LPS to induce ERK1/2 phosphorylation
on 2G5 and COS-7 cells is in agreement with previous results
showing that CD6 ligation induces MAPK activation (22) and
suggest that membrane CD6 is able to sense the presence of LPS
functional consequences will require future investigation.
It has been suggested that blockage of the main receptors that
mediate recognition of bacteria and their products may be a
suitable strategy for treating sepsis shock syndrome, which, at
were injected i.p. with a lethal dose of LPS (30 mg/kg) 1 h after i.p. adminis-
tration of sterile saline solution (n ? 26), rsCD5 (n ? 10), or rsCD6 (n ? 16) (25
P values were calculated. (B–D) Circulating levels of cytokines in LPS-
challenged mice. Plasma levels of TNF-? (B), IL-1? (C), and IL-6 (D) were
quantified by ELISA at different times after LPS injection. Data are expressed
as mean ? SEM. Statistical differences in the results were evaluated by the
two-tailed Student t test.*, Statistically significant difference (P ? 0.05).
Effect of rsCD6 and rsCD5 on survival rate and cytokine serum levels
Sarrias et al. PNAS ?
July 10, 2007 ?
vol. 104 ?
no. 28 ?
present, has a high mortality rate (up to 70% in septic patients)
(30, 31). Accordingly, strategies targeting or using several innate
immune receptors, such as activated protein C, TLR2, or CD14,
are being currently tested in animal models of septic shock and
also in clinical trials (32–34). Administration of a single dose (25
?g) of rsCD6 into mice 1 h before LPS challenge significantly
enhanced their survival rate (up to 70%) as compared with
rsCD5 or saline treatment (Fig. 6A) and concomitantly induced
a significant reduction in the levels of proinflammatory cyto-
kines TNF-?, IL-1?, and IL-6 in these mice (Fig. 6 B–D). The
bacterial aggregation data (Fig. 5A), together with the increase
of Ca2?-induced LPS aggregation in the presence of rsCD6 (Fig.
5B), suggest that rsCD6 may contribute to increase the size of
particles. This would facilitate particle clearance from the cir-
reduce subsequent inflammatory processes, which in cases such
as sepsis may even result in death.
It cannot be ruled out, however, that the antiinflammatory
effects of rsCD6 on LPS-induced septic shock may also result from
its inhibitory effects on different cell subsets involved in the
outcome of sepsis such as CD8 T cells and natural killer cells (35).
In fact, rsCD6 has been shown to inhibit human antigen-presenting
cell–T cell interactions (17, 36), and CD6 is known to be expressed
on natural killer cells (36). In any case, the septic shock data are of
great relevance because they constitute a therapeutic approach in
the prevention of LPS-induced septic shock.
In conclusion, the results of the present report illustrate the
unprecedented bacterial binding properties of the ectodomain
of CD6 and support its therapeutic potential for the interven-
tion of septic shock syndrome or other inflammatory diseases
of infectious origin. They also suggest that not only cells of the
innate immune system, but also T lymphocytes, may sense the
presence of bacterial components through CD6 as well as other
well known pattern-recognition receptors (e.g., TLRs), al-
though the functional consequences of such a recognition are
yet to be analyzed in depth. It can be hypothesized that, even
if its main role were the modulation of T cell activation and
differentiation signals, CD6 may have retained the ability to
recognize microbial components as an accessory property,
which is shared with other members of the ancient and highly
conserved SRCR-SF. This adds further evidence to the notion
that SRCR domains may have emerged as protein modules of
the innate immune system for recognition of pathogen-
associated molecular patterns.
Materials and Methods
Cells, Antibodies, and Reagents. The cell lines COS-7, Raji, K562,
and HUT-78 were from the American Type Culture Collection
(Manassas, VA). The CD5- and CD6-negative 2G5 cells (20)
were stably transfected with the pH?-CD6.P527stop and pH?-
CD6.wt constructs (12, 22). Cell growth conditions are detailed
in supporting information (SI) Methods. A full list of providers
of antibodies and reagents can be found in SI Methods.
Expression, Affinity Purification, and Biotin Labeling of Recombinant
Proteins. The recombinant proteins were expressed in HEK
293-EBNA and affinity-purified as reported (12, 37). Their
purity was assessed by SDS/PAGE and staining with Coomassie
blue. Protein biotinylation was performed with EZ-Link PEO-
maleimide-activated biotin (Pierce/Perbio Science, Cheshire,
U.K.) following the manufacturer’s instructions (8) and moni-
tored by Western blotting.
Bacterial Strains and Bacterial Binding Studies. The bacterial strains
used in this study are clinical isolates characterized by the
Department of Microbiology of the Hospital Clinic of Barcelona
using standard biochemical procedures. Bacterial growth con-
ditions are detailed in SI Methods. Binding of rsCD6 to bacteria
was studied following a method described previously (15), with
slight modifications (8).
Purification of Soluble CD6 from Human Serum. Soluble CD6 was
affinity-purified from 1 liter of human plasma pooled from
healthy blood donors obtained from the Blood Bank of the
Hospital Clinic de Barcelona as detailed in SI Methods.
LPS-Binding ELISAs. LPS purified from E. coli O55:B5, O111:B4,
or O26:B6 (Sigma, St. Louis, MO) was used to coat 96-well
microtiter plates (Nunc, Roskilde, Denmark) and assayed for
BSA, rsCD6, or nsCD6 binding as detailed in SI Methods.
Binding Assays of Soluble Proteins to FITC–Re-LPS. A fluorescent
Re-LPS derivative (FITC–Re-LPS) in which the phosphoeth-
anolamine group of Re-LPS was bound to FITC by a previously
described method (38). Fluorescence measurements were car-
ried out as previously described (8, 28, 29) and as described in
Bacteria and LPS Aggregation Assays. Bacteria aggregation assays
were performed as described (8). LPS aggregation induced by
rsCD6 was studied as before (29). For further details see SI
Flow Cytometry Assays. Cell-binding properties of soluble pro-
teins were assessed as described (13). Binding of LPS to
cell-surface CD6 was assessed by incubating cells with differ-
ent amounts of LPS–FITC from E. coli 0111:B4 (Sigma) in the
presence or absence of rsCD5, rsCD6, or BSA as detailed in
ERK1/2 Phosphorylation Assays. ERK1/2 phosphorylation was an-
alyzed by Western blot as described (22) (see SI Methods).
CD6 Immunoprecipitation. Immunoprecipitation of membrane
CD6 (mCD6) from HUT-78 T cells was performed and analyzed
by Western blotting as described (12).
LPS-Induced Endotoxic Shock. C57BL/6J mice (8 weeks old) were
injected i.p. with 25 ?g of saline solution, rsCD5, or rsCD6 1 h
before injection of an i.p. lethal dose of LPS from E. coli. For
further details see SI Methods.
Determination of Cytokine Serum Levels. The systemic release of
TNF-?, IL-1?, and IL-6 cytokines was determined by ELISA in
pooled serum samples as described in SI Methods.
We thank Dr. Jordi Vila for assistance with bacterial strains and Bele ´n
Sua ´rez for technical assistance. This work was supported by Ministerio
de Educacio ´n y Ciencia Grants SAF2004-03251 (to F.L.), BIO-2005-
01393 (to J.Y.), and SAF2006-04434 (to C.C.) and Instituto de Salud
Carlos III Grants PI05/1013 (to F.L.) and CB06/06/0002 (to C.C.).
M.-R.S. is supported by Fondo de Investigacio ´n Sanitaria Grant FIS
CP05/100. M.F., A.I., and I.G. are recipients of fellowships from Institut
d’Investigacions Biome `diques August Pi i Sunyer, Departament
d’Universitats, Recerca i Societat de la Informacio ´, and Ministerio de
Sanidad y Consumo–Institut d’Investigacions Biome `diques August Pi i
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