Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8110–8115, July 1999 Immunology

The mast cell tumor necrosis factor ␣ response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48

RAVI MALAVIYA*†,ZHIMIN GAO*, KRISHNAN THANKAVEL*, P. ANTON VAN DER MERWE‡, AND SOMAN N. ABRAHAM*§

*Department of Pathology and Microbiology, Duke University Medical Center, Durham, NC 27710; and ‡Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

Edited by Henry Metzger, National Institutes of Health, Bethesda, MD, and approved May 12, 1999 (received for review February 5, 1999)

ABSTRACT Mast cells are well known for their harmful fimbriae (7). This molecule has been shown to mediate bac- role in IgE-mediated hypersensitivity reactions, but their terial binding to mannosylated molecules on the surface of a physiological role remains a mystery. Several recent studies variety of host cells and in mucosal secretions (8, 9). Because have reported that mast cells play a critical role in innate FimH is expressed by many members of the Enterobacteriaceae immunity in mice by releasing tumor necrosis factor ␣ including well known pathogenic species such as E. coli, (TNF-␣) to recruit neutrophils to sites of enterobacterial Klebsiella pneumoniae, Serratia marcescens, and Salmonella infection. In some cases, the mast cell TNF-␣ response was typhimurium (7), mast cells have the potential to directly bind triggered when these cells directly bound FimH on the surface and respond to a wide range of enteric bacteria. There is of Escherichia coli. We have identified CD48, a glycosylphos- currently no information on the complementary FimH- phatidylinositol-anchored molecule, to be the complementary recognizing molecule(s) on the mast cell surface. Because of FimH-binding moiety in rodent mast cell membrane fractions. its relevance in identifying the molecular events leading to the We showed that (i) pretreatment of mast cell membranes with mast cell responses to bacteria, we sought to identify the antibodies to CD48 or phospholipase C inhibited binding of putative mast cell receptor for the FimH moiety of E. coli. FimH؉ E. coli,(ii) FimH؉ E. coli but not a FimH؊ derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) ؉ MATERIALS AND METHODS binding of FimH bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected Animals and Reagents. Male BALB͞c mice, 6–8 weeks old, with CD48 cDNA, and (iv) antibodies to CD48 specifically were purchased from Harlan–Sprague–Dawley. Animals were blocked the mast cell TNF-␣ response to FimH؉ E. coli. Thus, caged in groups of five in a pathogen-free environment, and CD48 is a functionally relevant microbial receptor on mast the experimental procedures were carried out in agreement cells that plays a role in triggering inflammation. with institutional guidelines. Fetal bovine serum was obtained from HyClone. BSA, Mast cells exhibit a remarkable capacity to release a battery of human gamma globulin, toluidine blue, hydrogen peroxide, ␣ ␣ inflammatory mediators when activated (1, 2). Since their D-glucose, -methyl D- mannopyranoside (Me Man), nitro discovery more than 100 years ago, the physiological role of blue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, phos- these cells, which are preferentially located at the host– pholipase C (PLC), and actinomycin D were purchased from environment interface, has been the subject of much debate. Sigma. Recombinant peptide-N-glycosidase F was obtained Recently, several laboratories have reported that mast cells are from Oxford Glycosystems (Rosedale, NY). Rat monoclonal central to protecting mice against lethal enterobacterial infec- antibody to mouse CD48 and mouse monoclonal antibody to tions through the release of various proinflammatory media- rat CD48 were purchased from Serotec, and rat monoclonal tors including tumor necrosis factor ␣ (TNF-␣), a potent antibody to mouse CD117 was purchased from PharMingen. In neutrophil chemoattractant (3–6). Cumulatively, these studies addition, a rat monoclonal antibody to mouse CD48 (7D1.G5) have established the vital role of mast cells in modulating host generated at the Sir William Dunn School of Pathology, defenses against infectious agents. Mast cells appeared to University of Oxford, was also used. contribute to the innate immune defenses because they were Bacterial Strains. E. coli ORN103(pSH2) is a recombinant activated by bacteria even in the absence of specific antibodies strain containing a plasmid, pSH2, that encodes all the to the pathogens. In one study, mast cell responses were necessary for the expression of functional type 1 fimbriae. E. elicited through bacterial activation of the host’s complement coli ORN103(pUT2002) is an isogenic FimH-minus derivative system because the in vivo inflammatory response to enter- (10). The bacterial strains were cultured in Luria broth con- obacteria was significantly reduced in complement-deficient taining chloramphenicol (50 ␮g͞ml). mice compared with wild-type mice (4). In another study, mast Cell Culture and Growth Conditions. Bone marrow mast cell activation in vivo appeared to be elicited through direct cells (BMMCs) were cultured from stem cells from the bone contact with cell surface molecules on Escherichia coli because mast cell release of inflammatory mediators was markedly This paper was submitted directly (Track II) to the Proceedings office. higher after exposure to wild-type bacteria than to an isogenic Abbreviations: TNF-␣, tumor necrosis factor ␣; GPI, glycosylphos- mutant deficient in FimH, a (3). FimH is a phatidylinositol; BMMC, bone marrow mast cell; CHO cells, Chinese 29-kDa mannose-binding lectin presented preferentially at the hamster ovary cells; DNP, 2,4-dinitrophenyl; PLC, phospholipase C; Me␣Man, ␣-methyl D-mannopyranoside; PVDF, poly(vinylidene di- distal tips of filamentous appendages on E. coli called type 1 fluoride). †Present address: Department of Allergy and Inflammatory Diseases, The publication costs of this article were defrayed in part by page charge Hughes Institute, 2665 Long Lake Road, St. Paul, MN 55113. §To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Pathology, Campus Box 3712, Duke University Medical Center, accordance with 18 U.S.C. §1734 solely to indicate this fact. Research Drive, Jones Building, Room 257, Durham, NC 27710. PNAS is available online at www.pnas.org. e-mail: [email protected].

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marrow of BALB͞c mice as described (11). The cells were to allow them to adhere to the bottom of the microtiter plates. grown in 25% WEHI-3 conditioned medium and used for The mast cell monolayers were then exposed to type 1 fim- experiments after 20 days in culture. Mast cells harvested from briated bacteria and the adherence assay performed as de- these cultures were Ͼ98% pure, as determined by toluidine scribed above. blue staining, and resembled the mucosal-type mast cells (11). Transfection of CHO Cells with CD48 cDNA. CHO cells RBL-2H3, a rat mast cell line, was a gift from Reuben P. were transfected with CD48 cDNA in the vector pKG5 using Siraganian (Laboratory of Microbiology and Immunology, the Transfectam reagent (Promega), and stably transfected National Institute of Dental Research, National Institutes of cells were derived by growth in G418-containing medium. Health). RBL-2H3 cells were cultured in 225-cm2 flasks con- Assays for TNF-␣. Monolayers of mast cells in 96-well trays taining Eagle’s essential medium supplemented with 20% FBS in serum-free RPMI-1640 medium containing 15 mM Hepes (12). Chinese hamster ovary (CHO) cells obtained from the were exposed to bacteria for 1 hr at 37°C or were sensitized American Type Culture Collection were cultured in Glasgow- with anti-dinitrophenyl (DNP) IgE and subsequently chal- modified Eagle medium (GMEM) supplemented with 10% lenged with DNP-BSA at 50 ng͞ml for 1 hr at 37°C as described FBS. The transfected CHO-CD48 cells were maintained in (16). After the incubation period, TNF-␣ in the cell-free GMEM supplemented with 10% FBS and G418 (Geneticin). supernatant was measured by a standard cytotoxicity assay (3). Flow cytometry analysis of cell lines was carried out. Briefly, cells were labeled with a mouse monoclonal antibody against RESULTS rat CD48 (diluted 1:10) followed by fluorescein isothiocya- nate-conjugated antibodies against mouse IgG (diluted 1:100). FimH-Expressing E. coli Recognize a 45-kDa Component of Cells were excited on FACScan with a 15-nW laser at 488 nm. Mast Cell Membranes. In our previous studies, we had used Results were presented as a histogram of the number of cells either IL-3-dependent mouse BMMCs or freshly isolated (10,000 per analysis) vs. the logarithmic scale of fluorescence peritoneal mast cells to study mast cell–enterobacteria inter- intensity. actions (3, 11, 17). However, we chose to isolate the FimH FimH͞FimC Complexes and Soluble CD48. Because of the receptor from the rat mast cell line RBL-2H3 because, unlike extreme sensitivity of the recombinant bacterial FimH to BMMCs or mouse peritoneal mast cells, these cells could be degradation in the bacterial periplasm, it is necessary to readily cultured to generate large numbers of cells (12). Our coexpress FimH with FimC, the fimbrial chaperone (13). The studies have shown that FimH-expressing E. coli bound in recombinant FimH and FimC form a highly stable comparable numbers to RBL-2H3 cells or to BMMCs (401 Ϯ complex in the bacterial periplasm that is readily isolated. 19 and 436 Ϯ 8 bacteria per 50 mast cells, respectively). FimC also serves to present FimH molecules in a functionally Moreover, the binding interactions were inhibitable by competent form (13). FimH–FimC complexes were isolated Me␣Man, indicating that the nature of the reaction with from the periplasm of the bacteria as described (13). Soluble FimH-expressing bacteria was likely to be identical in both mouse CD48 and rat CD48 (LRCD48) were obtained as types of mast cells. Our strategy to isolate the FimH receptor described (14, 15). Deglycosylation of mouse CD48 was in mast cells is summarized in Fig. 1. We reasoned that because achieved by treatment of the denatured glycoprotein with the FimH receptor contained mannose, we could use the peptide-N-glycosidase F as described by the manufacturer. mannose-binding Con A lectin to enrich for mannose- Ligand͞Lectin Blotting. After SDS͞PAGE and electro- containing molecules from the mast cell membrane prepara- phoretic transfer onto poly(vinylidene difluoride) (PVDF) tions. Pooled batch cultures of approximately 1011 cells were membranes, the membranes were blocked overnight with 3% prepared and a Triton X-100-soluble membrane fraction BSA in 10 mM Tris⅐HCl͞150 mM NaCl͞0.1% Tween 20 at pH (100,000 ϫ g fraction) was obtained as described (18). This 7.4 (TBST). Blots were incubated with 2 ϫ 108 biotinylated bacteria per ml (10) or 125I-labeled FimH͞FimC (50 ␮g͞ml) in TBST containing 1 mM CaCl2, 1 mM MgCl2, and 0.4 mM ␣ MnCl2 in the presence or absence of 100 mM Me Man at 4°C. After overnight incubation, the blots were washed three times with TBST. The blots treated with biotinylated bacteria were incubated with streptavidin linked to horseradish peroxidase (diluted 1:2,000) in TBST for 1 hr and developed with an appropriate substrate, whereas the blots incubated with 125I- labeled FimH͞FimC were developed by exposing to x-ray film for 48 hr. Bacterial Adherence Assay. Monolayers of BMMCs (5 ϫ 104 cells per well) in 96-well trays were blocked with 1% human gamma globulin for 1 hr, after which various concentrations of anti-CD48 or anti-CD117 were added. After 1 hr of incubation, unbound antibody was removed and then type 1 fimbriated E. coli suspended in serum-free RPMI-1640 medium was added at a bacteria͞mast cell ratio of 50:1. Unbound bacteria were gently rinsed off through three washes after 1 hr of incubation at 37°C. Triton X-100 (0.1%) was added to selectively solubi- lize the mast cells, and the total number of mast cell-associated bacteria was determined by standard viable counts as de- scribed (11). PLC Treatment of Mast Cells. BMMCs suspended in RPMI medium were treated with increasing concentrations of PLC (0.1–3.0 units͞ml) at 37°C for 1 hr. PLC was removed after repeated washing of the BMMCs with PBS, and the cells were resuspended in culture medium containing 1 ␮M actinomycin D. The cells were plated onto wells of a 96-well tissue culture FIG. 1. Strategy used to isolate and identify the putative FimH plate (5 ϫ 104 cells per well) and incubated overnight at 37°C receptor on mast cells. Downloaded by guest on September 24, 2021 8112 Immunology: Malaviya et al. Proc. Natl. Acad. Sci. USA 96 (1999)

membrane fraction was passed through a Sepharose-Con A microsequencing. A sequence of 12 amino acid residues in the affinity column to isolate candidate mannosylated compounds. N terminus were identified as 100% homologous to rat CD48 Because Con A also binds to glucose-containing molecules, we . CD48 is a glycosylphosphatidylinositol (GPI)-linked eliminated the membrane material that was binding to the molecule that has been reported to be present primarily on column by glucose residues by first washing with 100 mM cells of hematopoietic lineage (19, 20). Further confirmation glucose. None of the glucose-eluted material aggregated that the band recognized by FimH-expressing bacteria was FimH-expressing E. coli on glass slides, confirming that no indeed CD48 comes from the finding that a mouse monoclonal putative receptors were lost with this step. Next, 100 mM antibody directed at rat CD48 reacted with the 45-kDa band Me␣Man was used to elute all materials bound via their on a Western blot (Fig. 2, lane 7). It is also noteworthy that mannose residues. The resulting eluate was dialysed to remove when a whole cell lysate of BMMCs was probed with a rat Me␣Man, then concentrated, and subjected to SDS͞PAGE. monoclonal antibody (7D1.G5) to mouse CD48, an immuno- Many mannose-containing membrane components bound to reactive band corresponding to CD48 was seen in the prepa- the Con A column as evidenced by the large number of bands ration (Fig. 2, lane 8), confirming the presence of this molecule seen after staining of gels with Coomassie blue (Fig. 2, lane 1). on BMMCs. To identify the putative FimH-recognizing moiety among the Pretreatment of Mast Cells with PLC or CD48-Specific mannoside-eluted material, we electrophoretically transferred Antibody Results in Reduced Binding of FimH-Expressing E. the material after SDS͞PAGE onto PVDF membranes. The coli. Because GPI-linked moieties are cleaved off the surface immobilized material was then exposed to 125I-labeled recom- of host cells with PLC (21), we incubated BMMCs with binant E. coli FimH, which was presented in a complex with its increasing amounts of PLC before exposure to FimH express- chaperone, FimC (13). The chaperone is required to stabilize ing E. coli. PLC pretreatment of mast cells was found to inhibit the recombinant FimH protein and to present it in a function- bacterial binding in a dose-dependent fashion (Fig. 3A). More ally competent manner (13). As shown in Fig. 2, the FimH direct evidence implicating CD48 as the putative E. coli FimH probe specifically bound to a 45-kDa band in the absence (lane receptor comes from the observation that pretreatment of 2) but not in the presence (lane 3) of Me␣Man. Furthermore, when the blot was exposed to FimH-expressing E. coli ORN103(pSH2) and mutant FimH-deficient E. coli ORN103(pUT2002), only the former bound to the 45-kDa band (Fig. 2., lanes 4 and 5). The binding reaction of E. coli ORN103(pSH2) could be inhibited by 100 mM Me␣Man (Fig. 2, lane 6). These data indicate that E. coli FimH binds specifically to a 45-kDa mast cell membrane component and that the binding is via mannose residues on the membrane component because FimH is not glycosylated. The 45-kDa Moiety Recognized by FimH-Expressing E. coli on Mast Cells Is the Immune Recognition Molecule CD48. To determine the identity of the 45-kDa band, we purified the protein from the Con A-eluted fraction to homogeneity by FPLC using a Bio-Rad HQ column. The eluted proteins from this column were subjected to SDS͞PAGE and stained with Coomassie blue to visualize the 45-kDa band. The band of interest was transferred to PVDF membranes and subjected to

FIG. 3. Inhibition of mast cell association with FimH-expressing FIG. 2. Specific binding of recombinant FimH, FimH-expressing bacteria after pretreatment with increasing concentrations of PLC and bacteria, or CD48-specific antibody to CD48 in mast cell membrane antibody to CD48. (A) BMMC suspensions were pretreated with preparations. Lanes: 1, Me␣Man-eluted material from the Con A indicated concentrations of PLC for 1 hr and then PLC was removed. column after SDS͞PAGE and Coomassie blue staining; 2 and 3, PVDF The PLC treated cells were seeded in wells of a 96-well tissue culture membrane blots of Con A-eluted material probed with 125I-labeled plate in the presence of 1 ␮M actinomycin D. After overnight FimH–FimC complex in the absence or presence, respectively, of 100 incubation, bacterial adherence assays were carried out on these cells. mM Me␣Man; 4 and 5, PVDF membrane blots of Con A-eluted (B) Monolayers of BMMCs in a 96-well tissue culture plate were material probed with biotinylated FimH-expressing E. coli or biotin- coated with 1% human gamma globulin for 1 hr at 37°C. The ylated FimH-minus E. coli, respectively; 6, PVDF membrane blot of monolayers were then incubated with indicated concentrations of Con A-eluted material probed with biotinylated FimH-expressing E. antibody against either CD48 or CD117 (control) for 1 hr at 37°C. coli in the presence of 100 mM Me␣Man; 7, PVDF membrane blot of Bacterial adherence assays were then done, and the number of mast Con A-eluted material probed with rat CD48-specific mouse mono- cell associated bacteria was determined by standard viable counts on clonal antibody; 8, PVDF membrane blot of a whole cell lysate of agar plates. The data points represent the mean Ϯ SEM values BMMCs probed with rat monoclonal antibody to mouse CD48. obtained from three experiments. Downloaded by guest on September 24, 2021 Immunology: Malaviya et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8113

BMMCs with rat monoclonal antibodies to mouse CD48 inhibited the adherence of FimH expressing E. coli in a dose-dependent fashion, whereas rat monoclonal antibodies to mouse CD117 (c-), a well known mast cell membrane marker, did not (Fig. 3B). Thus, the putative FimH receptor on mast cells is the GPI-anchored moiety CD48. The Mannosylated Residues on CD48 Play a Critical Role in Mediating Binding to FimH-Expressing E. coli. We next examined the binding of FimH-expressing E. coli to soluble mouse CD48 in the presence and absence of Me␣Man. As shown in Fig. 4 (lanes 1 and 2), bacterial binding to CD48 was almost completely abolished in the presence of Me␣Man. That the glycosylated portion of CD48 was involved in binding was further confirmed by the observation that FimH-expressing bacteria failed to bind soluble CD48 after deglycosylation with peptide-N-glycosidase F (Fig. 4, lane 3). We also examined bacterial binding to an exclusively unprocessed form of soluble rat CD48 bearing high-mannose N-linked carbohydrates (15) and observed that bacterial binding was three times higher (as determined by densitometry) than binding levels seen with normally processed CD48 (Fig. 4, lanes 1 and 4). These experiments show that FimH-expressing E. coli can bind to recombinant CD48 in a cell-free system by specifically recog- nizing the mannosylated region of the molecule. Cell Surface Expression of CD48 on CHO Cells Increases Binding of FimH-Expressing E. coli. To determine whether expression of CD48 on cells that do not normally express this molecule can promote binding of FimH-expressing E. coli,we stably transfected CHO cells with the full-length cDNA en- coding rat CD48 (22). As shown in Fig. 5A, the flow cytometry analysis using mouse anti-rat CD48 as probe confirmed that virtually 100% of the transfected CHO cells expressed CD48, whereas none of the cells transfected with control cDNA FIG. 5. Induced expression of CD48 on CHO cells increases (encoding the C terminus of conglutinin) expressed CD48. binding of FimH-expressing E. coli.(A) Expression of CD48 on Further, we examined the transfectants for their capacity to transfected CHO cells assessed by flow cytometry analysis. The open bind FimH-expressing E. coli. The association of bacteria with peak represents control transfected CHO cells (with cDNA encoding these CD48-expressing cells was at least 4-fold higher than the the C terminus of conglutinin) and the solid peak represents CHO cells transfected with cDNA encoding rat CD48. The cells were labeled with number associated with CHO cells transfected with control CD48-specific mouse monoclonal antibody and fluorescein isothio- cDNA (Fig. 5B). Thus, CD48 molecules on transfected CHO cyanate-labeled second antibody. Similar results were obtained in cells are functional as FimH receptors. three experiments. (B) Binding of FimH-expressing bacteria to CD48- CD48 Is the Determinant on Mast Cells That Is Responsible expressing CHO cells and control CHO cells. CHO-cell-associated for Triggering the TNF-␣ Response to FimH-Expressing E. bacteria were quantitated by standard viability assays. The results are coli. We have already shown that CD48-specific antibody expressed as percent of control. The data points represent the mean Ϯ inhibits FimH-mediated bacterial binding to mast cells (Fig. SEM values obtained from three experiments. 3B). It is known that the early mast cell TNF-␣ response to bacteria plays a critical role in triggering the innate immune response to bacteria. We sought to demonstrate the role of CD48 in eliciting the mast cell TNF-␣ response to FimH- expressing bacteria after (i) blocking mast cell surface CD48 with CD48-specific antibody and (ii) removing CD48 from mast cell surfaces with PLC. Antibody to CD48, but not antibody to CD117, blocked mast cell TNF-␣ release in a dose-dependent manner (Fig. 6A). Further confirmation of the critical role of CD48 in the mast cell TNF-␣ response comes from the finding that pretreatment of BMMCs with increasing concentrations of PLC significantly reduced the mast cell’s capacity to release TNF-␣ after exposure to FimH-expressing E. coli (Fig. 6B). To ensure that either pretreatment did not, through some other mechanism, impair the mast cell TNF-␣ response, we examined the effect of both treatments on the mast cell’s ability to release TNF-␣ after stimulation by IgE FIG. 4. Binding of FimH-expressing E. coli to processed and unproc- and . Either pretreatment (tested at the highest con- essed recombinant CD48. Equal concentrations (5 ␮g) of processed centrations used in Fig. 6 A and B) did not reduce the mast cell (lanes 1 and 2), processed and deglycosylated (lane 3), and unprocessed TNF-␣ response to IgE and antigen (Fig. 6C). Thus, these ͞ (lane 4) soluble CD48 were subjected to SDS PAGE and then transferred observations provide definitive evidence that the mast cell onto PVDF membranes. The corresponding strips were exposed for 1 hr TNF-␣ response to FimH-expressing bacteria is mediated by to biotinylated FimH-expressing E. coli in the absence (lanes 1, 3, and 4) or presence (lane 2) of 100 mM Me␣Man. Bound bacteria were detected CD48 molecules present on the mast cell surface. Although by the addition of streptavidin-peroxidase and an appropriate substrate. there are likely to be other mannosylated moieties on the mast Similar results were obtained in two experiments. cell membrane capable of binding FimH-expressing E. coli, Downloaded by guest on September 24, 2021 8114 Immunology: Malaviya et al. Proc. Natl. Acad. Sci. USA 96 (1999)

appear to exist, one of which is mediated through other host opsonins, such as the iC3b fragment of complement. For example, the CR3 moiety on the surface of mast cells recog- nized S. typhimurium and the parasite Schistosoma mansoni after they were coated with iC3b component of complement (23, 24). The second recognition mechanism involves the direct interaction of bacterial surface molecules with comple- mentary molecules on the mast cell surface without a need for opsonins. So far, the best-described paradigm of these opso- nin-independent interactions involves enterobacteria express- ing type 1 fimbriae (17). FimH, a mannose-binding subunit, located preferentially at the type 1 fimbrial tips is the specific determinant recognized by the mast cell. Evidence of the role of the FimH moiety in mediating bacterial adhesion includes the finding that a FimH-negative E. coli mutant derivative exhibited limited mast cell binding but that the parental type 1 fimbriated (FimHϩ) E. coli bound avidly to mast cells (3, 17). Herein, we report that the complementary receptor on mast cells for bacterial FimH is CD48. This is based on the following evidence: (i) antibodies to CD48 specifically blocked binding of FimH-expressing bacteria to the surface of mast cells; (ii) FimH-expressing, but not FimH- deficient, bacteria mediated mannose- sensitive binding to recombinant CD48; and (iii) FimH-expressing E. coli bound in appreciably higher numbers to transfected CHO cells expressing CD48 compared with control-plasmid-transfected non-CD48-expressing CHO cells. CD48 is also a biologically active receptor because antibodies directed at CD48 specifically blocked the TNF-␣ response to bacteria. Thus CD48 is a physiologically relevant receptor for bacteria on mast cells. CD48 has been referred to as BCM1 in mice, OX 45 in rats, and Blast-1 in humans (14, 20, 22). The expression of CD48 is restricted to cells of hematopoietic lineage, particularly lym- phocytes, , , and mast cells. CD48 was discovered as a cell surface molecule expressed by human B in response to Epstein–Barr virus infection (25), but its physiologic role in the body is still unclear. Its interac- tion with CD2, particularly in rodents, suggests that it plays a central role in activation (26). Recently, its interaction FIG. 6. Blocking of the mast cell TNF-␣ response to FimH- with human epithelial cells was demonstrated, suggesting a expressing E. coli but not to IgE and antigen by pretreatment of role for CD48 in mediating interactions between lymphoid BMMCs with antibodies to CD48 and with PLC. (A) Human gamma cells and the epithelium (27). The CD48 ligand identified on globulin-treated monolayers of BMMCs were incubated with indicated epithelial cells has now been shown to be heparan sulfate (28). concentrations of antibody against either CD48 or CD117 (control) for ␣ Thus, the involvement of CD48 in bacterial recognition and in 1 hr at 37°C, and then bacteria were added to the wells. TNF- release ␣ from cell-free supernatants was measured after 1 hr. (B) BMMC triggering TNF- release in inflammatory cells represents a suspensions were pretreated with indicated concentrations of PLC for distinct function for this molecule. Rodent CD48 is mannosy- 1 hr. The PLC was removed by repeated washing, and the BMMCs lated and the primary structure of CD48 reveals several were seeded in wells of a 96 well tissue culture plate in the presence potential sites of (22). The importance of the of 1 ␮M actinomycin D to form monolayers. After overnight incuba- sugar moiety on CD48 and specifically its mannose residues is tion, the BMMC monolayers were exposed to FimH-expressing bac- ␣ indicated by the observation that binding of FimH-expressing teria and the TNF- released from cell-free supernatants was assayed E. coli to a highly glycosylated and unprocessed form of CD48 C ␣ after 1 hr. ( ) To examine the TNF- response of mast cells to IgE and was markedly higher than to processed CD48. Moreover, we antigen, untreated control BMMCs and BMMCs pretreated with PLC ␣ at 3 units͞ml or CD48-specific antibody at 30 ␮g͞ml were sensitized showed that Me Man inhibits the binding of both FimH- with anti-DNP IgE and subsequently challenged with DNP-BSA expressing E. coli and recombinant FimH to CD48. We have (antigen) at 50 ng͞ml for 1 hr at 37°C. TNF-␣ released from cell-free recently observed that human mast cells readily bind FimH supernatants was assayed thereafter. Data are the mean Ϯ SEM values expressing type 1 fimbriated E. coli but not its isogenic obtained from three experiments. FimH-minus mutant derivative (29). There is appreciable homology in covalent structure between human and rodent these studies show that CD48 is the biologically relevant CD48 (30). However, because it is the glycosylation pattern on receptor. CD48 rather than its protein configuration that is critical for bacterial recognition, it is difficult at this time to predict DISCUSSION whether or not CD48 will serve as the FimH receptor on human mast cells. Mast cells are found in relatively large numbers at the host– To date, several distinct receptors have been identified for environment interface, and recent studies have shown that E. coli type 1 fimbriae on different host cells, indicating these cells play a crucial role in microbial recognition and in considerable heterogeneity in the molecules mediating FimH modulating the innate immune response to these infectious recognition in these host cells. This is not surprising because agents. To effect this role, mast cells must possess the capacity it is the glycosylation pattern rather than the protein compo- to recognize microorganisms in the absence of microbe- nent of the receptor that is relevant to bacterial recognition. specific antibodies. Two separate recognition mechanisms These receptors include CD66 and CD67 on granulocytes (31), Downloaded by guest on September 24, 2021 Immunology: Malaviya et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8115

CD11b͞CD18 on neutrophils (32), uroplakin on uroepithelial 9. Ofek, I. & Sharon, N. (1990) Curr. Top. Microbiol. Immunol. 151, cells (33), and CD48 on (34). Moreover, a 91–113. number of constituents in mucosal secretions have been iden- 10. Thankavel, K., Madison, B., Ikeda, T., Malaviya, R., Shah, A. H., tified as receptors for type 1 fimbriated E. coli, including Arumugam, P. M. & Abraham, S. N. (1997) J. Clin. Invest. 100, Tamm–Horsfall protein in urine (35). CD48 joins a growing 1123–1136. Methods Enzymol. 253, class of GPI-anchored cell surface molecules that serve as 11. Malaviya, R. & Abraham, S. N. (1995) 27–43. receptors for microbes and their toxins. Some notable exam- 12. Okazaki, H., Zhang, J., Hamawy, M. M. & Siraganian, R. P. ples include CD14 for lipopolysaccharide (36), Thy-1 for (1997) J. Biol. Chem. 272, 32443–32447. aerolysin (37), and CD55 for echovirus, group B coxsackie 13. Jones, C. H., Pinkner, J. S., Nicholes, A. V., Slonim, L. N., viruses, and Dr-fimbriated E. coli (38). Engagement of these Abraham, S. N. & Hultgren, S. J. (1993) Proc. Natl. Acad. Sci. receptors by microbes or their products has been shown to USA 90, 8397–8401. trigger cellular responses (36–38). Engagement of CD48 by 14. Wong, Y. W., Williams, A. F., Kingsmore, S. F. & Seldin, M. F. FimH-expressing E. coli triggers the mast cell TNF-␣ response (1990) J. Exp. Med. 171, 2115–2130. because inhibition of FimH–CD48 interactions by a CD48- 15. Davis, S. J., Puklavec, M. J., Ashford, D. A., Harlos, K., Jones, specific antibody abrogates the mast cell TNF-␣ response. How E. Y., Stuart, D. I. & Williams, A. F. (1998) Prot. Engin. 6, CD48 and other GPI-anchored receptors, which are only 229–232. 16. Malaviya, R., Malaviya, R. & Jakschik, B. A. (1993) J. Biol. Chem. linked to the exoplasmic leaflet of the lipid bilayer of the 268, 4939–4944. plasma membrane actually transduce intracellular signals is 17. Malaviya, R., Ross, E. A., MacGregor, J. I., Ikeda, T., Little, J. R., not clear. However, many GPI-anchored moieties including Jakschik, B. A. & Abraham, S. N. (1994) J. Immunol. 152, CD48 are typically found in special glycolipid-enriched mi- 1907–1914. crodomains in the plasma membranes of cells (39). These 18. Lennartz, M. R., Wileman, T. E. & Stahl, P. D. (1987) Biochem. microdomains are rich in signaling molecules such as the J. 245, 705–711. heterotrimeric GTP-binding proteins that can potentially me- 19. Staunton, D. E. & Thorley-Lawson, D. A. (1987) EMBO J. 6, diate signal transduction from GPI-anchored proteins. G 3695–3701. proteins are involved in many signal-transduction pathways, 20. Yokoyama, S., Staunton, D., Fisher, R., Amiot, M., Fortin, J. J. including stimulation of adenylate cyclase, regulation of Ca2ϩ & Thorley-Lawson, D. A. (1991) J. Immunol. 146, 2192–2200. channels, stimulation of phospholipase A , stimulation of 21. Low, M. & Saltiel, R. (1988) Science 239, 268–275. 2 22. Killeen, N., Moessner, R., Arvieux, J., Willis, A. & Williams, A. F. phosphatidylinositol 3-kinase, and stimulation of PLC (40). A (1988) EMBO J. 7, 3087–3091. recent immunochemical study has shown that CD48 in lym- 23. Sher, A. (1976) Nature (London) 263, 334–336. phocytes is physically associated with GTP-binding proteins 24. Sher, A., Hein, A., Moser, G. & Caulfield, J. P. (1979) Lab. Invest. (40). Thus, engagement of CD48 could potentially trigger a 41, 490–499. mast cell response via a signaling pathway involving GTP- 25. Thorley-Lawson, D. A., Schooley, R. T., Bhan, A. K. & Nadler, binding proteins. Engagement of CD48 has also been shown to L. M. (1982) Cell 30, 415–425. activate Src family member tyrosine kinases, which are also 26. Davis, S. J. & van der Merwe, P. A. (1996) Immunol. Today 17, important effectors of signal transduction found associated 177–187. with glycolipid-enriched microdomains of the plasma mem- 27. Ianelli, C. J., Edson, C. M. & Thorley-Lawson, D. A. 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