The CD5 ectoExecutemain interacts with conserved fungal cell

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Philippa Marrack, National Jewish Medical and Research Center, Denver, CO, and approved November 26, 2008

↵1J.V. and R.F. contributed equally to this work. (received for review June 18, 2008)

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Abstract

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spα, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectoExecutemain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Weepptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectoExecutemain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptiExecuteglycan), and such binding is specifically competed by β-glucan but not by mannan. The Kd of the rshCD5/(1→3)-β-d-glucan phospDespise interaction is 3.7 ± 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding Preciseties of the CD5 ectoExecutemain is deduced from its protective Trace in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1→3)-β-d-glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

Keywords: β-glucansfungal sepsislymphocytesscavenger receptor

Pathogen recognition by the innate immune system relies on a limited number of fixed germline-encoded receptors, which have evolved to identify conserved microbial structures not shared by the host and essential for their survival, the so-called “pathogen-associated molecular patterns” (PAMPs) (1, 2). Examples of PAMPs are LPS from Gram-negative bacteria, lipotheichoic acid (LTA) and peptiExecuteglycan (PGN) from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and mannan from fungi. Several structurally and functionally diverse classes of pattern-recognition receptors (PRRs) exist that induce various host defense pathways. Protein Executemains involved in pattern recognition include, among others, the C-type lectin Executemain from dendritic cell (DC) lectins, the leucine-rich repeat from Toll-like receptors (TLRs), and the scavenger receptor cysteine-rich (SRCR) Executemains (2). The latter was Characterized on cloning of mouse type I class A macrophage scavenger receptor (SR-AI) (3). Sequence comparison with several other proteins, such as the sea urchin speract receptor, human and mouse CD5, and complement factor I, revealed the existence of a conserved, 100-aa-long motif characteristic of a new superfamily (SF) of protein receptors, named SRCR. This family is Recently composed of more than 30 different cell-surface and/or secreted proteins with representatives in most animal phyla, from low invertebrates to mammals (4). The SRCR-SF members are divided into 2 groups: Group A contains SRCR Executemains composed of 6 cysteines and encoded by 2 exons, whereas group B is composed of 8 cysteines and is encoded by a single exon. Recent structural data indicate, however, that both group A and B SRCR Executemains share a similar scaffAged (a central core formed by 2 antiparallel β-sheets cradling 1 α-helix), with the main Inequitys being observed at the connecting loops (5). This Position recalls that of the few other successful protein modules of the immune system from which evolution has settled and built a myriad of different proteins (e.g., Ig Executemain). The versatility of these conserved Executemains lies in the fact that key residues stabilizing the Executemain structure are conserved throughout evolution, whereas others can evolve freely (especially those at the external loops), giving rise to Distinguished functional diversity (6). Accordingly, there is not a unifying function reported for the SRCR Executemains despite their high degree of structural and phylogenetic conservation. Some of them have been involved in protein-protein interactions, with interaction of the CD6 lymphocyte receptor with CD166/ALCAM (7) and that of the CD163/M130 macrophage receptor with the hemoglobin-haptoglobin complex being the most well studied examples (8). A few members of both group A (i.e., SR-AI/II, MARCO, SCARA5) and group B (i.e., DMBT1, Spα, CD6) are known to interact with microbial surfaces. These interactions were initially mapped outside the SRCR Executemains (9), but recent evidence demonstrates the direct involvement of SRCR Executemains (10–13). However, whether pathogen scavenging is a general Precisety shared by all or only a selected group of SRCR-SF members still remains to be analyzed.

The lymphocyte receptors CD5 and CD6 are group B members of the SRCR-SF (4) that share Necessary similarities at both the structural and functional levels. They are encoded by contiguous genes thought to derive from duplication of a common ancestral gene (14) and are expressed on thymocytes, mature peripheral T cells, and B1a cells; the latter is a small subset of mature B cells responsible for the production of polyreactive natural antibodies, which is expanded in certain autoimmune and B-cell lymphoproliferative disorders (15). The extracellular Locations of both CD5 and CD6 are exclusively composed of 3 conseSliceive group B SRCR Executemains, which Display extensive amino acid sequence identity (5). The main Inequitys between CD5 and CD6 are found at their cytoplasmic Locations, which are devoid of intrinsic catalytic activity but contain several structural motifs compatible with a function in signal transduction (4). CD5 and CD6 are physically associated with the antigen-specific complex present on T and B cells (16, 17) and colocalize with it at the center of the immunological synapse (17, 18). Therefore, CD5 and CD6 are well positioned to modulate, either positively or negatively, the activation and differentiation signals generated by the antigen-specific receptor through still incompletely understood and complex signaling pathways (4, 7, 19, 20). This is likely achieved through engagement of their ectoExecutemains by different cell surface counterreceptors. Although it is well established that CD6 binds to CD166/ALCAM (7), a bona fide CD5 ligand has yet to be identified (4).

The bacterial binding capabilities of the CD5 and CD6 ectoExecutemains, both known to exist as soluble forms circulating in serum (21, 22), have been explored recently. The reported data indicate that soluble and membrane forms of CD6, but not of CD5, bind to Gram-negative and Gram-positive bacteria through recognition of LPS and LTA, respectively (13). The present report has extended these studies to fungal structures. Data provided herein indicate that the CD5 ectoExecutemain is well suited for the recognition of conserved components on fungal cell surfaces, namely β-glucans.

Results

The EctoExecutemain of Human CD5 Binds to Fungal Cells.

To study the microbial binding Preciseties of the extracellular Locations of CD5 and CD6 further (13), direct protein binding assays to fungi were performed. To this, biotin-labeled, affinity-purified, recombinant soluble proteins encompassing the 3 SRCR ectoExecutemains of human CD5 (rshCD5-b) or human CD6 (rshCD6-b) were incubated with whole fungal cells, and bound protein was then revealed by Western blotting. The rshCD5 and rshCD6 proteins have previously been Displayn to be indistinguishable (in apparent molecular mass, antibody reactivity, and cell binding Preciseties) from equivalent circulating forms present in normal human serum (13, 23). Fig. 1A Displays that rshCD5-b binds to both the saprophytic (S. pombe) and pathogenic (C. albicans, Weepptococcus neoformans) fungal cells tested, whereas rshCD6-b binds only to the saprophytic fungal cells. The binding of rshCD5-b was Executese dependent and saturable and was Distinguishedly facilitated by Ca2+ (Fig. 1B). According to previously reported data (13), either Dinky or no binding to Gram-negative (E. coli) or Gram-positive (S. aureus) bacteria was observed for rshCD5-b (Fig. 1C). This indicates that the extracellular Location of CD5 is well suited for recognition of fungal but not bacterial cell wall structures.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Interaction of the CD5 ectoExecutemain with whole fungal cells. (A) Biotin-labeled rshCD5 (rshCD5-b) or rshCD6 (rshCD6-b) (15 μg) was incubated with 106 S. pombe, C. albicans, or C. neoformans cells. Cell-bound protein was solubilized and run on SDS-PAGE. Detection of total and bound biotin-labeled proteins was performed by Western blot analysis by using HRP-streptavidin (SAv). (B) Binding of different amounts (1–20 μg) of rshCD5-b to C. albicans was analyzed as in A. The binding of 20 μg of rshCD5-b in presence of 5 mM EDTA is also Displayn. (C) Binding of a fixed amount (15 μg) of rshCD5-b or rshCD6-b to 108 E. coli or S. aureus bacteria is Displayn. (D) Culture supernatants from HEK 293-EBNA transfectants expressing individual ectoExecutemains (DI, DII, or DIII) of rshCD5 were incubated with 106 C. albicans or C. neoformans overnight at 4 °C. Unbound protein was washed off and precipitated with 10% (weight/vol) trichloroacetic acid. Unbound precipitated (NB) and cell-bound (B) proteins were analyzed by Western blot with a rabbit polyclonal anti-CD5 antiserum plus HRP-labeled sheep anti-rabbit Ig antiserum. (E) Fungal cell aggregation was assayed by incubating FITC-labeled C. albicans (106) overnight at 4 °C with 5–10 μg of BSA, rshCD5, or rshCD6 in the presence or absence of 20 μg of zymosan (ZYM), β-d-glucan (β-d-GLU), or mannan (MAN) as a competitor. Cells were then transferred onto glass slides and visualized by fluorescence microscopy. (Magnification: ×400.) Tot prot., total protein.

To identify which of the 3 SRCR Executemains of CD5 was involved in fungal interaction, further cell binding assays were performed by using culture supernatants from HEK 293-EBNA transfectants expressing individual soluble CD5 Executemains. As illustrated in Fig. 1D, all 3 individual SRCR Executemains of CD5 retained the ability to interact with fungal cell surfaces. This suggests that a conserved structural motif shared by all 3 SRCR Executemains is responsible for fungal scavenging.

Induction of Fungal Cell Aggregation by the CD5 EctoExecutemain.

Whether the presence of multiple binding sites on the CD5 ectoExecutemain would lead to fungal aggregation was further investigated by incubating FITC-labeled C. albicans cells with different concentrations of soluble unlabeled proteins (BSA, rshCD5, and rshCD6). As Displayn in Fig. 1E, rshCD5 induced Executese-dependent fungal aggregation, whereas neither rshCD6 nor BSA did. The same results were also observed when C. neoformans was assayed (data not Displayn). The rshCD5-induced fungal cell aggregation was significantly reduced in the presence of excess amounts of either zymosan or β-glucan but not mannan (Fig. 1E Bottom). This suggests that binding to and aggregation of fungal cells by rshCD5 is likely mediated through recognition of β-glucan, a structural component of fungal cell walls.

The CD5 EctoExecutemain Binds Directly to Conserved Components of Fungal but Not Bacterial Cell Walls.

The direct binding of the CD5 ectoExecutemain to purified microbial cell wall components was further analyzed by ELISA. In agreement with data Displayn in Fig. 1, Executese-dependent binding of rshCD5-b to plates coated with zymosan but not with LPS, LTA, or PGN was observed (Fig. 2A). As expected (13), rshCD6-b bound to LPS-, LTA-, PGN-, or zymosan-coated plates in a Executese-dependent manner (Fig. 2B). Fascinatingly, the absorbance values obtained for the interaction of zymosan with rshCD6-b were always lower than those obtained with rshCD5 (Fig. 2 A and B). This reinforces the suitability of the CD5 ectoExecutemain for scavenging fungal but not bacterial cell wall constituents, compared with the CD6 ectoExecutemain.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The CD5 ectoExecutemain binds to zymosan through recognition of β-d-glucans. (A) ELISA plates coated with BSA, zymosan (ZYM), LPS, PGN, or LTA were incubated with increasing amounts (0.01–2 μg) of rshCD5-b. Bound protein was detected with HRP-streptavidin (SAv). (B) Binding of rshCD6-b to BSA-, ZYM-, LPS-, PGN-, or LTA-coated ELISA plates was analyzed as in A. (C) Binding of rshCD5 to ZYM is competed by β-d-glucans. A fixed amount (2 μg) of rshCD5-b (Left) and rshCD6-b (Right) was incubated with ZYM-coated ELISA plates in the presence or absence of increasing amounts (0.01–20 μg) of unlabeled competitors (β-d-glucans, zymosan, mannan, or BSA). Bound protein was detected with HRP-SAv. (D) Binding of rshCD5 to whole fungal cells is competed by β-d-glucans. A fixed amount (15 μg) of rshCD5-b was incubated with 108 C. albicans or C. neoformans cell suspensions in the presence of increasing amounts (1–20 μg) of unlabeled competitors: Zymosan (S. cerevisiae), β-d-glucan (barley), β-1,3-glucan (E. gracilis), glucan (S. cerevisiae), and mannan (S. cerevisiae). Bound rshCD5-b was detected by Western blot analysis using HRP-SAv.

ELISA competition assays were next performed to determine which fungal wall component was responsible for the interaction with CD5. To this, the binding of a fixed amount (2 μg) of rshCD5-b to plastic-bound zymosan was competed with increasing concentrations of β-glucan, mannan, or zymosan. In accordance with results Displayn in Fig. 1, β-glucan and zymosan, but not mannan, were able to compete the binding of rshCD5-b to zymosan in a Executese-dependent manner (Fig. 2C). By Dissimilarity, the binding of rshCD6-b was only competed by zymosan (Fig. 2C).

The ability of different β-glucan–containing preparations to compete the binding of CD5 to fungal cell wall structures was further analyzed. The binding of a fixed amount of rshCD5-b (15 μg) to whole fungal cells was competed by increasing concentrations of β-glucan purified from barley, (1→3)-β-glucan from Euglena gracilis, and glucan from Saccharomyces cerevisiae as well as with zymosan or mannan (both from S. cerevisiae) used as positive and negative controls, respectively. As illustrated in Fig. 2D, all glucan preparations competed the binding of rshCD5-b to both C. albicans and C. neoformans in a Executese-dependent manner. This suggests that the interaction of the CD5 ectoExecutemain with fungi is likely mediated through recognition of β-glucan, a highly conserved and abundant constituent of fungal cell walls.

The interaction of CD5 with β-glucan was further analyzed by monitoring the changes in tryptophan fluorescence emission intensity of rshCD5 on excitation at 295 nm before and after addition of increasing amounts of (1→3)-β-d-glucan phospDespise. The spectrum of fluorescence emission of rshCD5 is characterized by an emission maximum at 335 nm [supporting information (SI) Fig. S1]. A glucan concentration-dependent increase of fluorescence intensity was observed for rshCD5 (Fig. S1A Left) but not for human serum albumin (Fig. S1B), reaching saturation at molar ratio of 1:1 (Fig. S1A Right). The apparent Kd was calculated and was 3.7 ± 0.2 nM.

Zymosan Binds to Membrane-Bound CD5 and Induces CD5-Mediated Activation of MAPK Cascade.

The interaction of membrane-bound CD5 with fungal cell wall constituents was next analyzed. To this, the binding of increasing amounts of FITC-labeled zymosan to 2G5, a Jurkat T-cell derivative selected for deficient expression of both CD5 and CD6 receptors (24), was investigated. As Displayn by Fig. 3A, fluorescence intensity of 2G5 cells transfected with CD5 (2G5-CD5.WT) was higher than that of untransfected cells. Competition binding experiments, as illustrated in Fig. 3B, Display that staining of 2G5-CD5.WT cells with a fixed amount of FITC-labeled zymosan (15 μg) was competed by increasing concentrations of unlabeled β-glucan and zymosan, but not mannan, in a Executese-dependent manner. These results suggest that CD5-expressing cells could sense the presence of conserved fungal cell wall constituents. Further evidence for the latter was obtained from activation of the MAPK signaling cascade in stable 2G5 transfectants expressing similar surface levels of either WT (2G5-CD5.WT) or cytoplasmic tail-less (2G5-CD5.K384Cease) forms of CD5 (25) (Fig. S2A). As Displayn in Fig. 3C, expoPositive to zymosan (40 μg/mL) induced time-dependent phosphorylation of both MEK and ERK1/2 in 2G5-CD5.WT cells but not in 2G5 cells. Zymosan-induced phosphorylation of both MEK and ERK1/2 was not observed in 2G5-CD5.K384Cease transfectants (Fig. 3C), which lack the most C-terminal 88 amino acids of CD5 (25). Similarly, abrogation of both MEK and ERK1/2 phosphorylation was observed in 2G5-CD5.WT cells exposed to zymosan in the presence of rshCD5 (Fig. S3). This indicates that activation of MAPK by zymosan in 2G5 cells depends on the expression of CD5 as well as on the integrity of its cytoplasmic Executemain.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Zymosan (ZYM) binds to membrane-bound CD5 and induces Executewnstream signaling events. (A) Increasing amounts (1–30 μg) of FITC-labeled ZYM were incubated with 2G5 cells either untransfected (Left) or transfected (Right) to express the WT membrane CD5 receptor (2G5-CD5.WT). Fluorescence intensity of stained cells was analyzed by flow cytometry. (B) 2G5-CD5.WT transfectants were stained with a fixed amount (15 μg) of FITC-labeled ZYM in the presence or absence of increasing amounts (1–30 μg) of ZYM, β-d-glucan (β-GLU), and mannan (MAN). Fluorescence intensity was analyzed by flow cytometry. (C) 2G5 cells (2 × 106) either untransfected or stably expressing WT (2G5-CD5.WT) or cytoplasmic tail-truncated (2G5-CD5-K384Cease) CD5 surface molecules were pulsed for 0, 5, 15, and 30 min with 40 μg/mL ZYM at 37 °C. Subsequently, cell solubilizates were analyzed by Western blot with anti-pERK1/2, anti-pMEK, and anti-cdk4 antisera plus HRP-labeled sheep anti-rabbit or anti-mouse Ig antisera. (D) HEK 293 cells either stably expressing TLR2 or not were transfected in transient for expression of WT (CD5.WT) or cytoplasmic tail-truncated (CD5.K384Cease) membrane CD5 forms. Cells were then pulsed for 24 h with 20 μg/mL ZYM in the presence or absence of rshCD5 (25 μg) and assayed for IL-8 secretion by ELISA.

Zymosan Induces CD5-Mediated Cytokine Release.

To explore the biological outcome of the interaction of membrane-bound CD5 with fungal cell wall constituents further, subsequent cytokine release was analyzed. Unfortunately, stimulation of both 2G5 and 2G5-CD5.WT cells did not result in significant cytokine release at different time points (data not Displayn). This unresponsiveness was observed following expoPositive to high concentrations of zymosan but also after expoPositive to potent T-cell specific stimuli, such as combinations of anti-CD3 and anti-CD28 mAb, thus indicating the likely existence of a blockade of cytokine release in 2G5 cells. In light of these observations, the membrane form of CD5 was expressed in a nonlymphoid mammalian cell system, the HEK 293 cells. Transient expression of WT (CD5.WT) and cytoplasmic tail-truncated (CD5.K384Cease) forms of CD5 was achieved on both the parental 293 cells and 293 cell transfectants stably expressing TLR2, a well-known receptor for zymosan (Fig. S2 B and C). Cells were then subjected to zymosan expoPositive (40 μg/mL) for 24 h, and IL-8 concentration was meaPositived. As Displayn in Fig. 3D, significant IL-8 release was observed for 293 cells expressing CD5.WT compared with either untransfected cells or cells expressing the cytoplasmic tail-less CD5.K384Cease. That release was significantly reduced by the presence of rshCD5. The IL-8 levels detected for 293-CD5.WT were similar to those observed for 293-TLR2 transfectants, which were used as a positive control. Moreover, coexpression of CD5.WT and TLR2 did result in additive Traces on IL-8 release following zymosan expoPositive. Once again, the presence of rshCD5 significantly reduced the zymosan-induced IL-8 release in both 293-TLR2 and 293-TLR2+CD5.WT transfectants. Taken toObtainher, the results indicate that the membrane form of CD5 senses the presence of fungal cell wall constituents, thereby initiating an independent signaling cascade resulting in cytokine release.

The Soluble CD5 EctoExecutemain Protects from Zymosan-Induced Septic Shock-Like Syndrome in Mice.

In vivo validation on the interaction of the CD5 ectoExecutemain with fungal constituents was obtained from the mouse model of septic shock-like syndrome induced by zymosan (26). In this model, administration of a single Executese of zymosan (500 mg/kg, i.p.) causes both aSlicee peritonitis and multiple organ injury within 18 h as well as increased mortality over a period of 12 days. As Displayn in Fig. 4, administration of a single i.p. Executese (25 μg) of rshCD5 1 h before zymosan challenge induced a significant reduction in toxicity score, peritoneal leukocyte count, IL-6 and IL-1β serum levels, and liver myeloperoxidase (MPO) activity at 18 h. Mouse survival was significantly increased (45% vs. 15%) for animals pretreated with rshCD5 compared with untreated controls. The Traces of rshCD5 on zymosan-induced septic shock-like syndrome were Displayn to be Executese dependent (Fig. S4). Contrary to rshCD5, the infusion of rshCD6 (25 μg, i.p.), before zymosan challenge, resulted in a nonsignificant reduction of the inflammation parameters analyzed (Fig. S5). Taken toObtainher, these data indicate that pretreatment of mice with rshCD5 prevents systemic inflammation induced by zymosan and unveils the therapeutic potential of rshCD5 for fungal septic shock. Accordingly, the anti-inflammatory Trace of rshCD5 was also evident when treatment was delayed for 1 or 3 h after zymosan administration (Fig. S6).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

rshCD5 protects from septic shock-like syndrome induced by zymosan in mice. rshCD5 pretreatment protects from zymosan-induced sepsis. The toxicity score, peritoneal total leukocyte count (103 cells/mm3), IL-6 and IL-1β serum levels, and liver MPO activity (mU/mg protein) of CD1 mice pretreated with BSA or rshCD5 (25 μg, i.p.) 1 h before infusion of zymosan (500 mg/kg, i.p.) were assessed at 18 h. The survival of mice from the same groups was monitored for 12 days.

Discussion

Fungal infections initiate mammalian immune responses through engagement of fungal-surface polysaccharides by a variety of cell-bound and/or soluble receptors, including mannose receptor, complement receptor 3 (CR3, CD11b/CD18), TLRs (TLR2, TLR4), collectins (SP-A, SP-D), pentraxin-3, and C-type lectins (dectin-1) (27). The present report Displays that CD5, a lymphoid member of the SRCR-SF, should be added to the list of receptors able to sense the presence of fungal cell wall components. The soluble CD5 ectoExecutemain not only binds to but aggregates several fungal species (saprophytic or pathogenic), and this is achieved through recognition of β-glucans, a major component of fungal cell walls. The relevance of this finding is highlighted by the fact that soluble CD5 prevents septic shock-like syndrome induced by the β-glucan-rich particle zymosan in mice.

β-glucans are Necessary cell wall components of mushrooms, seaweeds, yeast, and pathogenic fungi. Individual β-glucans are heterogeneous in terms of molecular weight, number of branches, and helical construction, but they basically consist of a backbone of polymerized β-(1→3)-linked β-d-glucopyranosyl units and β(1→6)-linked side chains. β-glucans can be recognized by a limited number of receptors, including CR3, lactosylceramide, scavenger receptors, and dectin-1 (27). The latter is considered the major β-glucan receptor on myeloid cells and is responsible for most biological Traces of β-glucans, such as TNF-α cytokine production by macrophages. Dectin-1 is a C-type lectin-like receptor highly expressed on neutrophils and inflammatory macrophages and, at lower levels, on resident macrophages, dendritic cells (DCs), B cells and, some T cells, although the functional significance of the latter remains to be explored (28).

The present report Displays that CD5 binds to zymosan, a polysaccharide particle from the cell wall of S. cerevisiae, which is the most commonly used β-glucan-containing experimental agent, although it also contains mannans, other glucans, and chitins. Fascinatingly, the binding of CD5 to zymosan and to different fungal specimens (C. albicans and C. neoformans) is competed by β-glucans prepared from different sources (barley, S. cerevisiae, and E. gracilis) but not mannan. One of the β-glucans used was β-1–3-glucan from E. gracilis, which is composed of a liArrive chain of glucose units without any branching. The apparent Kd of the interaction of rshCD5 with soluble (1→3)-β-d-glucan phospDespise was assessed and was 3.7 ± 0.2 nM, which is within the range of binding affinities (2.6 mM–2.2 pM) reported for the interaction of dectin-1 with (1→3)-β-d-glucan from different sources (29).

The fact that the soluble ectoExecutemain of CD5 not only binds but also aggregates fungal cells is of relevance, because aggregation is a common strategy used by components of the innate immune system to prevent pathogen dissemination and to facilitate pathogen clearance by phagocytes. Fungal aggregation is also indicative of the multivalency of the CD5 ectoExecutemain regarding fungal recognition. Indeed, each SRCR Executemain of CD5 retains the ability to bind to whole fungal cells. Thus, it is reasonable to speculate on the existence of a shared structural motif that is responsible for scavenging fungal cell wall components. Previous studies have Displayn the presence of 2 bacterial recognition motifs located on similar protein loops of the SRCR Executemains of MARCO (RXR) and DMBT1 (VEVLXXXXW) (10, 11). Available data Execute not implicate these 2 motifs in fungal recognition, and they are not fully conserved among the 3 SRCR Executemains of CD5. Thus, other yet unknown structural motifs may be responsible for the interaction of the SRCR Executemains of CD5 with fungi.

This study also Displays that zymosan, through binding to membrane-bound CD5, induces relevant signaling and biological Traces, such as activation of the MAPK cascade and IL-8 cytokine release. These 2 zymosan-induced phenomena are dependent on the integrity of the cytoplasmic tail of CD5. This confirms that CD5 is a surface receptor transducing signaling events and likely modulating cell activation and/or differentiation processes (19). Moreover, the zymosan-induced cytokine release was observed after expression of CD5 in nonlymphoid cells (HEK 293), and this would imply that different cell types may gain responsiveness to fungal cell wall components through acquisition of CD5 expression. CD5 is considered a lymphoid-specific receptor constitutively expressed on mature T and B1a lymphocytes (15), whose expression can be up-regulated under certain circumstances, such as chronic autoantigen stimulation in vivo of conventional B2 cells (30) or differentiation to cells with regulatory function (Tregs) (31). Moreover, extralymphoid expression of CD5 has been reported on enExecutethelial cells (32), macrophages (33, 34), and peripheral blood and vaginal DCs (35, 36). The ultimate reason for such expression is unknown, but it is tempting to speculate its possible involvement in pathogen detection. This is well exemplified for B1a cells, which are cells at the interphase of the innate and adaptive responses. B1a cells are responsible for the production of polyreactive natural antibodies recognizing either autoantigens or microbial components (15). Detection of anti-β-glucan antibodies in normal human and mouse sera has been previously reported (37, 38). Moreover, the protective Preciseties of CD5+ B cells in a rat vaginal candidiasis model have also been Displayn (39).

The binding of the CD5 ectoExecutemain to fungal wall components is underscored by the beneficial Traces observed in the mouse model of septic shock-like syndrome induced by zymosan. Pretreatment with a single i.p. Executese of rshCD5 induced significant reductions in toxicity score, serum levels of IL-6 and IL-1β, peritoneal and liver leukocyte infiltrates, and survival rate of mice challenged with a high Executese of zymosan. Moreover, the advantageous Traces of rshCD5 in zymosan-induced septic shock-like syndrome were still evident when its administration was delayed for 1 or 3 h after zymosan challenge. This suggests that rshCD5 should be considered as a potential therapeutic agent in fungal sepsis. The mechanistic basis for this protective Trace in vivo may rely on the neutralizing capability of zymosan particles by the soluble ectoExecutemain of CD5. In fact, the addition of rshCD5 inhibited in vitro phenomena directly triggered by zymosan (e.g., MAPK activation, IL-8 release). Moreover, rshCD5 also interfered with the IFN-γ release by peripheral T lymphocytes stimulated with plastic-bound anti-CD3 mAb either alone or in combination with zymosan (Fig. S7). This is in agreement with the previously Displayn inhibitory Trace of rshCD5 on CD3-mediated T-cell proliferative responses (18) and indicates that additional anti-inflammatory mechanisms may also account for the protective Preciseties of rshCD5 in fungal sepsis.

The present report also Displays the ability of the CD6 ectoExecutemain to recognize certain fungal structures of still unknown nature. This is not surprising, because high homology exists between the CD5 and CD6 lymphocyte receptors. However, Necessary Inequitys are found regarding bacterial recognition between the two molecules, and this also seems to be the case regarding fungal recognition. Compared with CD5, the soluble CD6 ectoExecutemain binds to a lesser spectrum (saprophytic but not pathogenic) of fungal species, it binds to fungal cell wall structures distinct from the highly conserved β-glucans and mannans, it binds to zymosan with lower avidity as deduced from ELISAs, and it Executees not induce significant protection against zymosan-induced septic shock-like syndrome. Taken toObtainher, this indicates that the ectoExecutemains of CD5 and CD6 are complementary regarding pathogen recognition: whereas CD6 is well suited for bacterial (both Gram-negative and Gram-positive) detection, CD5 is better adapted for fungal binding. This implies that, by coexpressing CD5 and CD6, lymphocytes could increase their sensing potential for microbial products in the extracellular milieu. This agrees with accumulating evidence Displaying the expression of different PRRs on lymphocytes (28, 40). Although the functional consequences of PRR ligation by microbial components on lymphocytes is a matter of Recent study, available evidence indicates that lymphocytes Retort directly to microbial products through PRRs and that this modulates their functions (40–44). Recently, a model of TLR-mediated control of Treg function during pathogen challenge has been proposed in which TLR2 ligands rapidly increase the host's adaptive immunity by expanding Traceors and also by attenuating suppressive activity of Tregs (42, 45); in the process, Tregs also expand and recover their suppressive activity when the infection has subsided, in time to limit potential autoimmunity from overactivated Traceors (43, 46). Intriguingly, CD5 has been found up-regulated on Tregs (31) as well on anergic T (47, 48) and B (30) cells. Whether simultaneous binding of CD5 (and/or CD6) to microbial products collaborates in the TLR-mediated control of adaptive immune responses needs further investigation.

In conclusion, the present study unveils the unpDepartnted fungal binding Preciseties of lymphoid members of the SRCR-SF, namely, CD5 and, to a lesser extent, CD6. Whether this is a general Precisety shared by other members of the SRCR-SF still remains to be explored. Moreover, the present results add further evidence to the notion that the SRCR Executemains may have emerged as protein modules of the innate immune system and have evolved to built-in different structural motifs for recognition of different PAMPs. Finally, the present results also support the therapeutic potential of the CD5 ectoExecutemain for the intervention of septic shock syndrome or other inflammatory disorders of fungal origin.

Materials and Methods

Constructions.

A detailed description of the cloning and expression procedures of constructs coding for soluble (rshCD5, rshCD6, rshCD5.DI, rshCD5.DII, and rshCD5.DIII) and membrane-bound (CD5.WT and CD5.K384Cease) proteins can be found in SI Appendix.

Cells.

The source and the growth conditions of the cell lines and transfectants used in this study (2G5, HEK 293-EBNA, HEK 293, and HEK 293-TLR2) are detailed in SI Appendix.

Purification and Biotin Labeling of Recombinant Proteins.

Individual ectoExecutemains of CD5 were used as unFragmentated serum-free culture supernatants, whereas rshCD5 and rshCD6 were affinity-purified as Characterized in SI Appendix. Protein biotinylation was performed with EZ-Link PEO-maleimide–activated biotin (Pierce/Perbio Science) following the Producer's instructions.

Bacterial and Fungal Binding Studies.

The bacterial and fungal cells used were clinical isolates. Cells were suspended to a final density of 1010 bacteria or 108 fungi per milliliter, and binding to recombinant proteins was analyzed in the presence or absence of competitors as Characterized in SI Appendix. ELISA binding assays were performed on 96-well plates (Nunc) coated with 20 μg of LPS, LTA, PGN, or zymosan (all from Sigma) and then incubated with biotin-labeled BSA, rshCD5, or rshCD6 in the presence or absence of cAged competitors as detailed in SI Appendix. Fluorescence assays to determine the binding characteristics of rshCD5 to soluble (1→3)-β-d-glucan phospDespise were Executene as Characterized in SI Appendix.

Fungal Aggregation Assays.

Aggregation of FITC-labeled fungal cells (106) in the presence or absence of competitors was performed as Characterized in SI Appendix.

Flow Cytometry Analysis.

2G5 cells (2 × 105) either untransfected or stably expressing CD5.WT were stained with different amounts of FITC-labeled zymosan in the presence or absence of increasing amounts of cAged competitors as detailed in SI Appendix.

Cytokine Assays.

Culture supernatants of HEK transfectants pulsed with 20 μg/mL zymosan for 24 h were assayed for IL-8 by ELISA (BD OptEIA, Human IL-8 ELISA Set; BD Biosciences) following the Producer's instructions. ELISA for mouse IL-6 and IL-1β serum level determination was performed according to the Producer's protocols (Quantikine Immunoassay; R&D Systems).

MAP Kinase Assays.

Cell lysate samples from 24-h serum-starved cells (2 × 107) stimulated with zymosan (40 μg/mL) were assayed for ERK1/2 and MEK phosphorylation as Characterized in SI Appendix.

Zymosan-Induced Septic Shock-Like Syndrome.

Male CD1 mice were subjected to zymosan-induced septic shock-like syndrome as previously Characterized (26). A single i.p. Executese of rshCD5, rshCD6, or BSA was given before or after zymosan challenge (500 mg/kg). For further details, see SI Appendix.

Acknowledgments

We thank J. Vila (Department of Microbiology, Hospital Clinic of Barcelona) for providing bacterial and fungal specimens and B. Suárez, C. Serra, N. Climent, M. Antón, and C. Astasio for technical assistance. This work was supported by Spanish Ministry of Education Grants SAF2004–3251 and SAF 2007–62197 (to F.L.) and SAF2006–04434 (to C.C.), Spanish Research Network on Infectious Diseases Grant RD06/0008/1013 (to F.L.), and National Institutes of Health Grant GM53522 (to D.L.W.). M.-R.S. is the recipient of a fellowship from FonExecute de Investigación Sanitaria (CP05/100).

Footnotes

2To whom corRetortence should be addressed at: Servei d'Immunología, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. E-mail: flozano{at}clinic.ub.es

Author contributions: J.V., R.F., C.C., J.Y., and F.L. designed research; J.V., R.F., O.C., M.F., and R.M. performed research; O.C., D.L.W., and C.C. contributed new reagents/analytic tools; J.V., R.F., O.C., M.F., R.M., D.L.W., C.C., J.Y., and F.L. analyzed data; and J.V., R.F., M.-R.S., D.L.W., C.C., J.Y., and F.L. wrote the paper.

Conflict of interest statement: This work is the subject of a patent application (ES200801860).

This article is a PNAS Direct Submission.

See Commentary on page 1303.

This article contains supporting information online at www.pnas.org/cgi/content/full/0805846106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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