In this study, we demonstrate potent regulatory function of the murine killer cell inhibitory receptor-like molecules, paired Ig-like receptors (PIRs) or p91, using chimeric receptors expressed on the rat basophilic leukemia cell line RBL-2H3. One of the chimeras, which has the transmembrane and cytoplasmic domain of PIR-B fused to the extracellular portion of type IIB receptor for IgG, was able to inhibit the type I receptor for IgE-mediated degranulation response upon coaggregation. This chimera also suppressed cytoplasmic Ca2+ mobilization in the presence and absence of calcium ion in the extracellular medium. Tyrosine to phenylalanine point mutations at the third and fourth immunoreceptor tyrosine-based inhibitory motif-like sequences of PIR-B attenuated the inhibitory effects on degranulation and on cytoplasmic Ca2+ mobilization, indicating the important role of these tyrosines for the delivery of negative signal. In contrast, the cross-linking of another chimeric receptor composed of the type IIB receptor for IgG extracellular portion and the transmembrane and short cytoplasmic sequence of PIR-A elicited Ca2+ mobilization and degranulation. These results indicate that PIR molecules may regulate cellular functions both positively and negatively.
In recent years, considerable progress has been made in understanding the receptors responsible for activating and inhibiting immune cell functions. For example, type III receptor for IgG (FcγRIII)4 is an activating FcR composed of a ligand-binding α subunit and either a CD3ζ or FcRγ chain homodimer or a ζγ heterodimer containing one or three sets of immunoreceptor tyrosine-based activation motif (ITAM) (1); this homodimer or heterodimer recruits an src homology 2 (SH2)-containing protein tyrosine kinase Syk after phosphorylation of the tyrosine residues in the ITAM and transmits the activation signal to the cell interior (2, 3, 4). In contrast, a type II FcR for IgG (FcγRIIB) is known to suppress activation, proliferation, and Ig production of B cells when cross-linked with B cell receptor (BCR) using intact anti-Ig (5, 6, 7). The mechanism of this suppression can be explained in terms of the phosphorylation of a tyrosine residue in its cytoplasmic region in the immunoreceptor tyrosine-based inhibitory motif (ITIM) (8) followed by the recruitment of an SH2-containing inositol phosphatase (SHIP) (9, 10, 11) upon coligation with BCR.
The analogous mode of regulation is used in the human killer cell inhibitory receptor (KIR) and killer cell activatory receptor (KAR), which recognize MHC class I on the target cells and inhibit or activate NK cell-mediated cytolysis (12, 13, 14, 15, 16, 17), respectively. For the delivery of the inhibitory signal by KIR, SH2-containing tyrosine phosphatase (SHP)-1 recruitment after tyrosine phosphorylation in its ITIM has been shown to be critical (18, 19). KARs do not contain any ITIMs or express a single charged amino acid residue within their transmembrane domain as do several activating-type receptors such as FcγRIIIα chain. Recently, KAR was shown to be associated with a novel set of phosphorylated polypeptides called KAR-associated polypeptides (20) or to a disulfide-bonded homodimer, DAP12, containing an ITAM (21).
p91 (22) or paired Ig-like receptor (PIR) (under agreement with Dr. Max D. Cooper (University of Alabama at Birmingham, Birmingham, AL), we hereafter use this standardized nomenclature) (23) is a family of murine type I transmembrane glycoproteins belonging to the Ig superfamily, which was recently cloned by us and others. Northern blot analysis and RT-PCR revealed that these molecules are expressed in B cells, macrophages, and mast cells (22, 23, 24) but not in T cells or NK cells (22, 24), although the physiologic ligand(s) for p91/PIR are not known. One isoform, PIR-B (or p91A) (22, 23, 24) has six Ig-like domains in the extracellular portion and four ITIM-like sequences in the cytoplasmic region. The considerable similarities of the PIR-B molecule to human KIR (25) and mouse gp49B1 (26, 27) suggest an inhibitory role for this molecule when cross-linked with an activatory-type receptor by a currently undefined ligand(s). In contrast to PIR-B, PIR-A molecules (23) (or p91B, Ref. 24; or p91D, GenBank accession no. AF055896) do not contain any ITIMs in their cytoplasmic portion or express a charged amino acid residue, arginine, within their transmembrane domain, as is the case for other stimulatory-type receptors such as the FcγRIIIα chain; the FcγRIIIα chain associates with a CD3ζ or FcRγ chain homodimer or a ζγ heterodimer containing one or three sets of ITAM, suggesting its potent activating function.
To address whether the cytoplasmic regions of PIR are potentially stimulatory or inhibitory in the context of cellular activation, we made chimeric receptors, which consist of PIR-B or PIR-A transmembrane and cytoplasmic regions fused to the extracellular portion of FcγRIIB, and transfected them into the rat basophilic leukemia cell line RBL-2H3. This report describes the inhibitory nature of PIR-B, the importance of tyrosines in the third and the fourth ITIM-like sequences found in the cytoplasmic portion for the delivery of negative signal, and the stimulatory role of PIR-A.
Materials and Methods
Cell line and Abs
The rat basophilic leukemia cell line RBL-2H3 (28) was obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and maintained in DMEM containing l-glutamine, 20 μM 2-ME, penicillin, streptomycin, and 8% FBS. Biotinylated anti-mouse FcγRII/III mAb 2.4G2 (29) was purchased from PharMingen (San Diego, CA). Anti-SHIP antiserum was a generous gift of Dr. K. Mark Coggeshall (Ohio State University, Columbus, OH). Anti-SHP-1 or anti-SHP-2 IgG were products of Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was obtained from Sigma (St. Louis, MO). Biotinylated anti-rat MHC class I mAb (anti-RT 1.A) was purchased from Cedarlane Laboratories (Hornby, Canada).
For FcγRIIB-PIR-B chimeric receptor construction, a restriction site-added PIR-B cDNA fragment was produced by PCR using a forward primer, 5′-GCGGGCCCCATATGTACCTGAAGGCT-3′ (harboring nucleotide residues 1912–1929) (22), and a reverse primer, 5′-ATGGGCCCTTCCCAGAGTCCCTTGGA-3′ (harboring a reverse strand of residues 2559–2576), and ligated into the ApaI site of mouse FcγRIIB in the expression vector pcEXV-3 (30). All mutations were introduced by the PCR method using the following primers: 5′-AGGATGTGACCTTTGCCCAGCTG-3′ (corresponding to nucleotide residues 2369–2391 with a substitution at 2381) (22) and 5′-ACAGCTGGGCAAAGGTCACATC-3′ (a reverse strand of residues 2371–2392 with a substitution at 2381); 5′-GAGCCCAGTGTATTTGCTACTCTG-3′ (residues 2458–2481 with a substitution at 2471) and 5′-CCAGAGTAGCAAATACACTGG-3′ (a reverse strand of residues 2462–2482 with a substitution at 2471). The isolation of cDNA clones for PIR-A (or p91D) by screening a B10.A mouse cDNA library prepared from thioglycolate-elicited macrophages was described previously (24). The PIR-A cDNA was subcloned into pUC19 or pBluescript (Stratagene, La Jolla, CA) and sequenced using a Cy5 AutoRead sequencing kit and an ALFexpress DNA sequencer (both from Pharmacia Biotech, Uppsala, Sweden). A homology search and alignment of the nucleotide and amino acid sequences was performed as described previously (22, 24). For FcγRIIB-PIR-A chimera, the PIR-A cDNA was produced using a primer pair of 5′-GAGGGCCCCACACAATGGAGAATCTCAT-3′ (harboring residues 1873–1892; GenBank accession no. AF055896) and 5′-AAGGGCCCATCAGCTTTATTTCCCAGCG-3′ (harboring a reverse strand of residues 2030–2050) and ligated into the ApaI site of FcγRIIB in the pcEXV-3. RBL-2H3 cells were electroporated at 250 V/975 μF with the linearized chimera construct (20 μg) plus pSV2-neo (1 μg), selected with 100 μg/ml of G418 (Life Technologies, Grand Island, NY), and cloned by the limiting dilution method. The surface expression of the chimeric construct on each clone was monitored by flow cytometry on a FACSCalibur (Becton Dickinson, San Jose, CA) with biotinylated 2.4G2 and phycoerythrin (PE)-avidin (PharMingen).
Proteins bound to the tyrosine-phosphorylated peptide were detected by Western blotting as described previously (24). Briefly, 5 μl of streptavidin (SA)-coupled beads (Sigma) were incubated with 5 μg of biotinylated, phosphorylated, or nonphosphorylated peptides suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin, 2 mM NaVO4, 50 mM NaF, and 10% glycerol) at 4°C for 1 h followed by 1 mM of d-biotin solution blocking at 4°C for 1 h. Exponentially growing RBL-2H3 cells were washed with PBS and solubilized in lysis buffer on ice. Supernatant of cell lysates was incubated with the above beads at 4°C for 1 h. Precipitates on beads were washed three times in lysis buffer and resuspended in SDS sample buffer. Denatured and reduced samples were separated with 7.5% SDS-PAGE and transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA). Blots were incubated with anti-SHIP, anti-SHP-1, or anti-SHP-2 Abs in 5% skim milk solution in 0.3% Tween 20-Tris-buffered saline followed by HRP-conjugated anti-rabbit IgG. The signals were detected by enhanced chemiluminescence (Amersham, Little Chalfont, U.K.).
Cell priming and stimulation for degranulation
A total of 2 × 106 RBL-2H3 cells were first primed with biotinylated anti-trinitrophenyl (TNP) IgE mAb (0.2 μg/ml) (31) and/or biotinylated 2.4G2 (1 μg/ml) at 37°C for 10 min and washed. TNP-OVA or SA was used as cross-linking reagent. Briefly, primed 5 × 105 RBL-2H3 cells were incubated with 10 ng/ml of TNP-OVA or 1/1000 diluted SA-HRP complex (Amersham) solution in 200 μl of RPMI 1640 medium supplemented with 1% FBS at 37°C for 30 min.
Assay of degranulation
The intensity of degranulation was measured by the β-glucuronidase activity in the culture supernatant (32). Culture supernatant of the stimulated RBL-2H3 cells was added to an equal volume of 5 mg/ml p-nitrophenyl β-d-glucuronide solution in 1 M acetate buffer (pH 4.0) and reacted at 37°C for 4 h. A total of 60 μl of the reaction was then added to 120 μl of 0.2 M glycine buffer (pH 11.7) containing 1% SDS. Absorbance of 415 nm was measured, and the percentage of degranulation was calculated using the following formula, where spontaneous release represents the culture without stimuli, and maximum release represents the culture with 0.09% Triton X-100:
Percentage of degranulation = ([OD415 of test well − OD415 of spontaneous release]/[OD415 of maximum release − OD415 of spontaneous release]) × 100.
Analysis of cytoplasmic Ca2+ mobilization
A total of 5 × 106 RBL-2H3 cells were incubated with 2 μM of fura-2/acetoxymethyl ester and biotinylated anti-TNP IgE for 30 min at 37°C and then reacted with biotinylated 2.4G2 or anti-rat MHC class I for 10 min at room temperature. An appropriate loading of washed cells (1.5 × 106) was suspended in 2 ml of PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 or in HEPES-buffered saline with 1 mM EGTA and then stimulated with 10 μg of SA. Cytoplasmic Ca2+ mobilization was monitored at a 510-nm emission wavelength excited by 340 nm and 360 nm using a fluorescence spectrophotometer (model F-4500, Hitachi, Tokyo, Japan). Calibration and calculation of Ca2+ concentration was performed as described previously (33).
As the first step toward elucidating the inhibitory or activating function of PIR molecules, we constructed chimeric receptors (Fig. 1 A), since Abs to the extracellular portion of PIR are not available. The chimeric molecule with the PIR cytoplasmic portion and the FcγRIIB extracellular domain enables us to cross-link PIR/Fcγ RIIB chimeric molecules as well as to coaggregate the cytoplasmic portions of PIR to any known receptors. In this study, we focused on FcεRI-mediated signal as a target of PIR-mediated modulation, because PIR-A and PIR-B have been shown to be expressed on mast cells (22, 23, 24).
Association of phosphatases to ITIM-like sequences in vitro
To determine which of the cytoplasmic ITIM-like sequences of PIR-B could act as an inhibitory motif within RBL-2H3 cells, point-mutated PIR-B chimeras were prepared and tested. We have shown previously that tyrosine-phosphorylated but not nonphosphorylated synthetic peptides matching each of the third and fourth ITIM-like sequences of PIR-B can associate with SHP-1 and SHP-2 from A20 lymphoma and macrophage cell extracts, whereas those peptides corresponding to each of the first and second sequences cannot (24). As shown in Figure 2, this was also true for the RBL-2H3 cell extract. SHIP association with the phosphotyrosyl peptides harboring each of the third and fourth ITIM-like sequences was also observed, but its binding to the latter sequence was less significant. Based on these findings, we introduced tyrosine to phenylalanine point mutations into the third, fourth, and both ITIM-like sequences of PIR-B chimeric receptor, designated PIR-B-Y3F, PIR-B-Y4F, and PIR-B-YWF chimeras, respectively (Fig. 1,A). In addition, we made a chimeric construct containing a transmembrane and a short cytoplasmic domain of PIR-A, which does not harbor ITIM sequence (PIR-A chimera, Fig. 1,A). Figure 1 B shows the expression level of each chimeric receptor on transfected RBL-2H3 cells. We used these clones, which had almost equivalent expression levels of the chimeric molecules, for the additional experiments.
Inhibition of degranulation response by PIR-B
Figure 3 shows a suppressive effect of PIR-B chimera on FcεRI-mediated activation. TNP-OVA or SA was used as cross-linking reagent. We confirmed that aggregation of FcεRI by the addition of TNP-OVA to the sensitized cells was capable of releasing granule enzyme (Fig. 3, left panels). Receptor aggregation by SA after biotin-IgE plus biotin-2.4G2 sensitization resulted in suppressed degranulation as compared with sensitization with biotin-IgE alone (Fig. 3, right panels). The degranulation efficiency of FcεRI aggregation by SA paralleled that by TNP-OVA stimulation. FcγRIIB, which has been shown to have an inhibitory effect when cross-linked with BCR (5, 6, 7, 8, 34), FcγRIIA, FcεRI, and TCR (35), also showed the same suppressive effect in the same condition as that described above. Interestingly, two point mutants, the PIR-B-Y3F and PIR-B-Y4F chimeras, showed reduced suppression compared with the intact PIR-B chimera. Thus, tyrosine residues in the third and fourth ITIM-like sequences are required for the inhibitory nature of PIR-B.
Inhibition of cytoplasmic Ca2+ mobilization by PIR-B
The inhibitory effect of the PIR-B cytoplasmic portion was also observed in cytoplasmic Ca2+ mobilization experiments. The Ca2+ response in the PIR-B chimera transfectant was suppressed when the chimera receptor was coaggregated with FcεRI (Fig. 4,A). The same transfectant showed minimum inhibition when sensitized with biotin-IgE and biotin-anti-MHC class I, suggesting that the inhibition seen in the PIR-B chimera and FcεRI coaggregation was not due to a nonspecific dilution effect on FcεRI receptor aggregation (Fig. 4,B). PIR-B-Y3F and PIR-B-Y4F mutant transfectants showed decreased cytoplasmic Ca2+ responses, but these responses were less significant than that in the intact PIR-B chimera (Fig. 4 C). An almost complete abrogation of inhibitory effect was observed in a PIR-B-YWF chimera transfectant, confirming the importance of both of these tyrosine residues in the third and fourth ITIM-like sequences for negative signaling. It should be noted that the inhibition of cytoplasmic Ca2+ mobilization by PIR-B chimera transfectant was not affected by the presence of EGTA in the external medium, which is in contrast to the case for FcγRIIB inhibition; this finding indicates that PIR-B suppresses the mobilization of internal calcium as opposed to external calcium.
Stimulatory function of PIR-A
To address whether the noninhibitory-type PIR molecules, PIR-A, may function as activating receptors, we made a chimeric receptor composed of an extracellular portion of FcγRIIB and a transmembrane and short cytoplasmic tail of PIR-A (Fig. 1,A). We successfully obtained a transfectant expressing the PIR-A chimeric receptor on RBL-2H3 cells (Fig. 1,B). Again, SA was used for stimulation after sensitization with biotin-IgE and/or biotin-2.4G2. Interestingly, cross-linking the receptors with biotin-2.4G2 and SA elicited Ca2+ mobilization without coaggregating the receptors with FcεRI (Fig. 5,A). Coaggregation of PIR-A chimera with FcεRI induced a prolonged Ca2+ response when compared with that by FcεRI aggregation (Fig. 5,A). Furthermore, we found that cross-linking of PIR-A chimera elicited a substantial degranulation response without FcεRI coaggregation (Fig. 5 B). Thus, the PIR-A transmembrane and short cytoplasmic tail have a potent stimulatory effect on cytoplasmic Ca2+ mobilization followed by degranulation response.
In this study, we have demonstrated, through the use of chimeric receptors composed of the mouse FcγRIIB extracellular portion fused to the transmembrane and cytoplasmic portion of PIR, that PIR-B and PIR-A have inhibitory and stimulatory functions, respectively, in RBL-2H3 cells. Although a rat FcγRII analogue of the mouse FcγRIIB isoform has been identified in RBL-2H3 cells (36), the potential interference of rat FcγRIIB is unlikely, because the rat anti-mouse FcγRII mAb 2.4G2 does not cross-react with rat FcR (29).
It is important to clarify which part of the cytoplasmic sequence of PIR-B molecule can deliver such negative signaling. We verified that tyrosine residues in the third and fourth ITIM-like sequences are required for the inhibitory nature of PIR-B (Figs. 3 and 4). Recently, we observed that PIR-B was tyrosine-phosphorylated upon pervanadate treatment of COS cell transfectant (24). Together with these results, the most plausible explanation would be that both of these tyrosines are phosphorylated upon receptor coaggregation and recruit SH2-containing phosphatases as noted in Figure 2.
SHIP-dependent signal is clearly distinguished from the SHP-1- and SHP-2-dependent ones by the Ca2+ dependency in the extracellular fluid (37). FcγRIIB-mediated inhibition, which is dependent upon SHIP, is sensitive to the presence of EGTA in the medium, whereas KIR inhibition, which is dependent upon SHP-1, is resistant to Ca2+ chelation by EGTA (37). As shown in Figure 4,A, the cytoplasmic Ca2+ mobilization profile of FcγRIIB transfectant in the presence of EGTA did not change regardless of the stimulation with anti-FcγRIIB Ab, which is in agreement with the previous observation for SHIP dependency. This result supports previous reports indicating that FcγRIIB engagement inhibits Ca2+ influx (8, 38), whereas KIR acts on the release of intracellular Ca2+ (39). Importantly, the inhibition of cytoplasmic Ca2+ mobilization by PIR-B chimera transfectant was not affected by the presence of EGTA in the external medium, which is in contrast to the case for FcγRIIB inhibition (Fig. 4 A). The inhibition by PIR-B chimera in the presence of EGTA suggests the physiologic importance of SHP-1 and SHP-2 in the signal transduction of PIR-B inhibition in contrast to FcγRIIB signaling. Thus, the in vitro observations that phosphotyrosyl ITIM peptides bound SHP-1, SHP-2, and SHIP from RBL-2H3 cells might not accurately reflect the situation in vivo.
In agreement with the observation that the point mutations at tyrosine residues in the third or fourth ITIM-like sequences attenuated the inhibitory nature of PIR-B (Figs. 3 and 4), an almost complete abrogation of inhibitory effect was observed in a PIR-B-YWF chimera transfectant (Fig. 4,C). However, two PIR-B-YWF clones showed incomplete abrogation of the suppressed cytoplasmic Ca2+ mobilization response (data not shown). This inconsistency might arise from the six- to eightfold higher expression levels of the PIR-B-YWF mutant on the latter two transfectants than that on the first one shown in Figure 1. Thus, we cannot exclude the possibility that the first and second ITIM-like sequences and/or unidentified signaling motif(s) in the PIR-B cytoplasmic region also serve for the negative signaling.
The PIR-A transmembrane and short cytoplasmic tail have a potent stimulatory effect on the cytoplasmic Ca2+ mobilization and degranulation response (Fig. 5). It is plausible that the PIR-A chimeric receptor might be associated with signaling molecule(s) harboring a stimulatory motif such as ITAM and being expressed intrinsically in RBL-2H3 cells, because the PIR-A cytoplasmic 13-aa residues, WRSHRQTHPVAGN (in one-letter code), do not seem to have any known stimulatory motifs other than the serine residue that might be phosphorylated by protein kinase C. Recent findings that KAR on NK cells in humans associates with DAP12 (21), which has an ITAM sequence (21), support this notion. The nature of associating molecules of PIR-A remains to be clarified.
Although the specific ligand(s) of PIR have not been identified, Cosman et al. (40) recently cloned a novel Ig superfamily glycoprotein, leukocyte Ig-like receptor-1, by expression cloning using the viral MHC class I molecule as a probe; this glycoprotein turned out to be identical with Ig-like transcript-2 (41), one of the human homologues of the PIR family (23, 24). More recently, Borges et al. (42) and Colonna et al. (43) have reported that some but not all of the leukocyte Ig-like receptor/Ig-like transcript family members bind MHC class I molecules. Taken together with the above findings, the polymorphisms in the PIR-B extracellular domains between mouse strains and the chromosomal location of PIR (23, 24) suggest MHC class I as one of the plausible candidates for ligand of PIR, although the biologic significance of this ligand-receptor interaction in B cells, macrophages, or mast cells is not clear. Such interaction might play an important role in regulating these cells to react with autologous cells or Ags.
Note added in proof. Recently, Maeda, A., et al. (J. Exp. Med., In press) reported the stimulatory nature of PIR-A.
We thank Dr. K. M. Coggeshall (Ohio State University, Columbus, Ohio) for anti-SHIP antiserum and Dr. T. Kurosaki (Kansai Medical University, Osaka, Japan) for helpful discussions.
This work is supported by research grants from the Ministry of Education, Science, Sports, and Culture of Japan and from Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST) (to T.T.).
Abbreviations used in this paper: FcγRIIB and FcγRIII, type IIB and type III low-affinity receptors for IgG, respectively; BCR, B cell receptor; FcεRI, type I receptor for IgE; KAR, killer cell activatory receptors; KIR, killer cell inhibitory receptors; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; PIR, paired Ig-like receptors; SH2, src homology 2; HRP, horseradish peroxidase; SA, streptavidin; SHIP, SH2-containing inositol phosphatase; SHP, SH2-containing tyrosine phosphatase; PE, phycoerythrin; TNP, trinitrophenyl.