Human Mast Cells Functionally Expressed on Skin-Derived Riib, Is

The Journal of Immunology

Fc␥RIIa, Not Fc␥RIIb, Is Constitutively and Functionally Expressed on Skin-Derived Human Mast Cells1

Wei Zhao,2* Christopher L. Kepley,2† Penelope A. Morel,‡ Lawrence M. Okumoto,* Yoshihiro Fukuoka,† and Lawrence B. Schwartz3†

␥ The expression of Fc R by human skin-derived mast cells of the MCTC type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by ␤ ␣ ␥ release of -hexosaminidase, PGD2, leukotriene C4 (LTC4), IL-5, IL-6, IL-13, GM-CSF, and TNF- .Fc RIIa was consistently detected along with Fc␧RI at the mRNA and protein levels; Fc␥RIIc was sometimes detected only by RT-PCR; but Fc␥RIIb, ␥ ␥ ␥ Fc RI, and Fc RIII mRNA and protein were not detected. Fc RIIa-specific mAb caused skin MCTC cells to degranulate and ␣ ␧ secrete PGD2, LTC4, GM-CSF, IL-5, IL-6, IL-13, and TNF- in a dose-dependent fashion. Fc RI-specific mAb caused similar

amounts of each mediator to be released with the exception of LTC4, which was not released by this agonist. Simultaneous but independent cross-linking of Fc␧RI and Fc␥RIIa did not substantially alter mediator release above or below levels observed with

each agent alone. Skin MCTC cells sensitized with dust-mite-speciﬁc IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals. The Journal of Immunology, 2006, 177: 694–701.

eceptors for IgG mediate important regulatory and effec- Later release of newly formed cytokines and chemokines further tor functions within a variety of cell types, including contributes to this inflammatory process. In addition to their well- R mast cells. Three classes have been described, Fc␥RI established role as the effector cells for allergic inflammation, mu- (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16), each having dif- rine studies indicate mast cells participate in the innate immune ␥ ferent affinities for IgG. Fc RI, a high-affinity receptor (IgG3 Kd response against bacteria and viruses (2–4) and also against certain Ϫ ϳ10 9 M), is composed of an ␣-chain that binds IgG (IgG3 Ͼ parasites (5, 6). Rodent mast cells play key roles in the pathogen- 1Ͼ4 Ͼ 2), and the ␥-chain dimer, each of which contains an ITAM esis of non-IgE-mediated hypersensitivity disorders (7–9) involv- motif (4) that transmits signals leading to cell activation and me- ing Fc␥RIIIa that is naturally expressed on their surfaces and diator release. Fc␥RII is a 40-kDa monomeric glycoprotein with which responds to IgG immune complexes by causing secretion of Ն Ϫ7 low affinity for human IgG (IgG3 Kd 10 M). Three genes in mediators. Although Fc␥RIII expression has not been reported on by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd. humans, FCGRIIA, FCGRIIB, and FCGRIIC, and one in mice, human mast cells, functional Fc␥RI is transiently induced by FCGRIIB, encode the different subtypes. FCGRIIA and FCGRIIC IFN-␥ (10). In mice activating Fc␥RIIIa and Fc␧RI molecules are each encode an ITAM, while FCGRIIB encodes an ITIM (1), counterbalanced by inhibitory Fc␥RIIb (11, 12). When Fc␥RIIb is ␥ Ͼ ϭ Ͼ which attenuates ITAM signaling. Fc RIIa binds IgG3 1 2 coaggregated with Fc⑀RI, degranulation is attenuated (13) due to ␥ ␥ Ͼ ϭ Ͼ 4, while Fc RIIb and Fc RIIc bind IgG3 1 4 2. FCGRIII the ITIM domain (14), which recruits the phosphatase SHIP1 (15). ϭ Ͼ ϭ Ն Ϫ6 binds IgG3 1 2 4 with low affinity (IgG3 Kd 10 M) Mice lacking Fc␥RIIb have augmented allergic responses (16, 17). and is derived from two genes. The attenuating function of Fc␥RIIb has been postulated to ex- Mast cells are ubiquitously located in tissues where they initiate plain how allergen immunotherapy might work: allergen injections and propagate inflammatory diseases. They are uniquely equipped

http://classic.jimmunol.org induce allergen-specific IgG in the presence of allergen-specific to initiate type I hypersensitivity reactions through their activation IgE, and the resultant IgG–allergen–IgE complexes co-aggregate by multivalent Ags that cross-link IgE-bound Fc␧RI on the surface Fc␧RI and Fc␥RIIb. CD32 expression has been reported on human of mast cells, which induces the release of preformed mediators basophils and skin mast cells (18) but not on lung mast cells (19). such as histamine and newly generated mediators such as PGD . 2 In support of this pathway, a bispecific anti-IgE/anti-Fc␥RII Ab was constructed that inhibited allergen–IgE-mediated activation of

† Downloaded from Departments of *Pediatrics and Internal Medicine, Virginia Commonwealth Univer- human basophils and cord blood-derived mast cells (20). But it sity, Richmond, VA 23298; and ‡Department of Immunology, University of Pitts- was not clear whether this bispeciﬁc Ab acted through an ITIM burgh, Pittsburgh, PA 15213 motif, competed with IgE for binding to Fc␧RI, and/or interfered Received for publication October 24, 2005. Accepted for publication April 14, 2006. with the aggregation of IgE by Ag. A potentially therapeutic bispe- The costs of publication of this article were defrayed in part by the payment of page ciﬁc human Fc␥-Fc␧ chimera, called GE2, was constructed and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. shown to inhibit class switching to IgE and IgE production by ␧ 1 This work was supported in part by National Institutes of Health Grants R01- human B cells (21) and mediator release from Fc RI-expressing AI27517 (to L.B.S.) and K08-AI057357 (to W.Z.), by a grant from Philip Morris dendritic cells (22). GE2 also attenuated allergen-dependent acti- USA and Philip Morris International (to L.B.S.), and by the Food Allergy and Ana- vation of human cord blood-derived mast cells and peripheral phylaxis Network (to C.L.K.) blood basophils, and allergen skin tests in monkeys (23, 24). How- 2 W.Z. and C.L.K. contributed equally to this manuscript. ever, whether tissue-derived mast cells express Fc␥R capable of 3 Address correspondence and reprint request to Dr. Lawrence B. Schwartz, Virginia Commonwealth University, P.O. Box 980263, Richmond, VA 23298. E-mail address: inducing inhibitory signals, similar to rodent mast cells and human [email protected] cord blood-mast cells, is not clear.

The current study examines human skin-derived mast cells (25) viously (36). Southern blot analysis of PCR products was performed for expression of Fc␥R, and shows that they express functional using a digoxygenin-UTP (Boehringer Mannheim) labeled RS91-46 Fc␥RIIa, but not Fc␥RI, Fc␥RIIb, Fc␥RIIc, and Fc␥RIII, and con- probe as described previously (37). sequently can release mediators in an IgG-dependent, IgE-inde- Flow cytometry pendent manner. Cells were recovered by centrifugation at 800 ϫ g at 4°C, washed with PBS/1%BSA, and blocked for 30 min at 4°C with a 1/500 dilution of Materials and Methods normal human serum. The cells were washed and incubated with the in- Abs and reagents dicated Ab (10 ␮g/ml) for1hat4°C. After Ab labeling, the cells were Ј washed and incubated with a 1/100 dilution of F(ab )2-FITC-goat anti- Anti-Fc␧RI␣ mAb (22E7) (26) was a gift from Dr. J. Kochan (Hoffman-La mouse Ab (BD Pharmingen) for 30 min at 4°C. After three washes, cells Roche, Nutley, NJ), anti-Fc␥RIIb and anti-Fc␥RIIc mAb (41H16) were were resuspended in 400 ␮l of PBS. The mean intensity of fluorescence from Dr. B. M. Longenecker (Edmonton, Canada) (27), and rabbit poly- was determined for at least 10,000 cells using a FACScan flow cytometer clonal Abs were raised against the cytoplasmic tail of Fc␥RIIb (Ab163) (BD Biosciences). MOPC31C, nonspecific IgG, was used as a negative (28) and of Fc␥RIIa (Ab260) (29) from Dr. Clark Anderson (Ohio State control. All experiments were performed at least three times. University, Columbus, OH). Anti-Fc␥RIIa, anti-Fc␥RIIb, and anti-Fc␥RIIc ␥ mAb (2E1) (30) (Santa Cruz Biotechnology); anti-Fc RIIa mAb (IV.3) Cell activation (31), anti-Fc␥RIIa, anti-Fc␥RIIb, anti-Fc␥RIIc mAb (AT10) (32), and anti- Fc␥RI mAb (32.2) (Medarex); nonspecific mouse IgG1 mAb (MOPC31C), Cultured skin mast cells (0.5 ϫ 105 cells) were washed and activated in anti-Fc␥RIII mAb (3G8) mAb, and soybean trypsin inhibitor (SBTI)4 (Sigma- AIM-V with Abs at the concentrations indicated. In some experiments, Aldrich); anti-Kit mAb (YB5.B8) (Immunotech); recombinant Der p2 (33), freshly dispersed Percoll-enriched skin mast cells were activated with these mouse (Fab)-human (Fc) chimeric Der P2-specific IgE (2B12) (34) and Abs. For degranulation and lipid mediator measurements, activation was IgG1 (␣DpX) (35) mAbs (Indoor Biotechnologies); 4-hydroxy-3-nitrophe- stopped after 30 min by adding three volumes of ice-cold PBS. The cells nylacetyl (NP)-BSA (ϳ24 NP moieties per BSA molecule; Biosearch were centrifuged at 1000 rpm for 10 min at 4°C. Supernatants were trans- Technology); and NP-specific mouse (Fab)-human (Fc) IgE (MCA333S) ferred into a separate tube. Cell pellets were resuspend in PBS, sonicated and IgG2 (MCA334B) (Serotec) were obtained as indicated. in a Branson sonifier (model 350; power 5, 50% pulse cycle ϫ 4 pulses) and microfuged. For cultured skin mast cells where purities Ͼ95%, ␤-hex- Culture of human skin mast cells osaminidase was assayed by measuring release of p-nitrophenol from the substrate p-nitrophenyl N-acetyl-␤-D-glucosaminide as described (38). Ab- All study protocols involving human tissues were approved by the Human sorbance values were read at 405 nm. For the freshly isolated skin mast Studies Internal Review Board at Virginia Commonwealth University cells where purities were typically ϳ5%, tryptase levels were measured in (Richmond, VA). Surgical skin samples were obtained from Virginia Com- the releasates and retentates by ELISA using G4-biotin and B12 anti- monwealth University Medical Center, the Cooperative Human Tissue tryptase mAbs as described (39). In each case, degranulation was calcu- Network of the National Cancer Institute, or the National Disease Research lated as a percentage of release values using the following formula: Interchange. Skin-derived mast cells were prepared as described (25). After removing s.c. fat by blunt dissection, residual tissue is cut into 1- to 2-mm stimulated release % release ϭ ϫ 100 fragments and digested with type 2 collagenase (1.5 mg/ml), hyaluronidase stimulated ͑release ϩ retained͒ (0.7 mg/ml), and type 1 DNase (0.3 mg/ml) in HBSS for2hat37oC. The dispersed cells were collected by filtering through a no. 80 mesh sieve and To determine cytokine production, human skin mast cells were washed, resuspended in HBSS containing 1% FCS and 10 mM HEPES. Cells were suspended at 106 cells/ml in AIM-V medium containing SBTI (100 ␮g/ml) resuspended in HBSS, layered over a Percoll cushion, and centrifuged at and activated in 24-well plates by incubation with anti-Fc␧RI (22E7) mAb by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd. ϫ 700 g at room temperature for 20 min. Nucleated cells were collected at 1 ␮g/ml for 24 h as described (40). from the buffer/Percoll interface, while erythrocytes sediment to the bottom of the tube. Cells enriched by Percoll density-dependent sedimentation ␧ ␥ were resuspended at a concentration of 1 ϫ 106 cells/ml in serum-free Fc RI–Fc RII coaggregation AIM-V medium (Life Technologies) containing 100 ng/ml of recombinant To examine Fc␧RI–Fc␥RII coaggregation, mast cells were sensitized with human stem cell factor (SCF) (a gift from Amgen). Skin mast cells were Der p2-specific IgE (10 ␮g/ml), Der p2-specific IgG1 (10 ␮g/ml) (each split into separate wells every 4–5 days. Total cell numbers and viabilities recognizing a noncompeting epitope (41) or both for at least2hat37°C. were assessed by trypan blue staining. Cultures of skin-derived mast cells This human IgG1 mAb bound to the mast cell surface under our experi- ϳ were maintained for up to 3 mo and were 100% mast cells. Alternatively, mental conditions as determined by flow cytometry (data not shown). Cells freshly dispersed, Percoll-enriched mast cells were labeled with anti- were washed and activated with 5 ␮g/ml Der p2 that had been aggregated Fc␧RI-␣ and anti-CD117 mAbs (5 ␮g/ml), and then with FITC-labeled 3

http://classic.jimmunol.org using a (BS )-coupling procedure according to the manufacturer (Pierce), Ј o Ն anti-mouse F(ab )2 at 4 C. Labeled cells were puriﬁed to 95% by sorting or with buffer or mAb controls. in a MoFlo high-performance cell sorter (Cytomation) and subjected to Immune complexes were generated with IgE and IgG anti-NP mAbs and Western blotting for detection of different forms of CD32. NP-BSA. Stock solutions of NP-BSA (0.13 ␮g/ml) and Ig (8.7 ␮g/ml) were prepared and used to challenge skin-derived mast cells. Reactions Gene expression were stopped after 30 min and analyzed for degranulation as above. RNA extracted from skin-derived mast cells in culture with SCF was Lipid and cytokine measurement

Downloaded from reverse-transcribed, labeled, and hybridized to Affymetrix HG-U133A and HG-U133B GeneChips by the DNA Microarray Section of the Nu- Cysteinyl-leukotriene levels in releasates were measured with an enzyme cleic Acids Core Facility at Virginia Commonwealth University as rec- immunoassay (EIA) for leukotriene C4 (LTC4)/D4 (LTD4)/E4 (LTE4) (Am- ommended by the manufacturer. The average signal intensity of genes ersham Biosciences). For this EIA, the cross-reactivity between LTC and determined to be expressed by the detection of cDNA was normalized 4 LTD4 is 100%; LTC4 and LTE4 is 70%; and LTC4 and LTB4 is 0.3%. to 500. Concentrations were determined according to the manufacturer’s instruc- For RT-PCR total cellular RNA was extracted using the MicroFast- tions with a lower limit of detection of 15 pg/ml. Track Method (Invitrogen Life Technologies) from 1 ϫ 106 mast cells A PGD2-MOX EIA kit (Cayman Chemical) was used to measure PGD2 or from the peripheral blood leukocytes shown previously to express levels according to manufacturer’s instructions. PGD from fresh samples Fc␥RIIa, Fc␥RIIb, and Fc␥RIIc isoforms. RNA was concentrated by 2 was first converted into PGD2 methoxime to prevent degradation. The ethanol precipitation, resuspended in diethylpyrocarbonate-treated wa- lower limit of detection was 10 pg/ml. Ϫ ␮ ter, and stored at 70°C. cDNA was synthesized from 20 lofRNA Cytokines were measured using sandwich ELISAs in 384-well plates as ␥ using a first strand cDNA kit (Pharmacia Biotech). Fc RII isoforms described (40). Purified and biotinylated mouse or rat mAbs specific for were specifically amplified using primers and conditions described pre- each cytokine and standard recombinant cytokines were purchased from BD Biosciences as follows: IL-5, rat JES1-39D10/JES1-5A10; IL-6, rat MQ2–13A5/MQ2–39C3; IL-13, rat JES10–5A2/mouse B69–2; GM-CSF, 4 Abbreviations used in this paper: SBTI, soybean trypsin inhibitor; LT, leukotriene; rat BVD2-23B6/BVD2-21C11; and TNF-␣, mouse MAB1/MAB11. Lower NP, 4-hydroxy-3-nitrophenylacetyl; EIA, enzyme immunoassay. limits of sensitivity were 16 pg/ml. 696 Fc␥RIIa ON HUMAN MAST CELLS

Table I. Gene expression of IgG and IgE receptors on skin-derived mast cells using Affymetrix gene chips

Gene Product Expression Signal

Fc␧RI␣ 3260 Fc␧RI␤ 2979 Fc␧RI␥ 6453 Fc␥RI (CD64) Aa Fc␥RIIa (CD32a) 133 Fc␥RIIb (CD32b) A Fc␥RIIc (CD32c) A Fc␥RIII (CD16) A Fc␧RII (CD23) A

a A, absent.

Statistical analysis One-way ANOVA was used to compare data among different treatment FIGURE 2. Surface expression of mast cell Fc␥RII. Mast cells were groups. If signiﬁcant differences were detected, a Dunnett test was then incubated at 4°C with mAbs against Fc␥R, Fc␧RI, and Kit as described, followed by FITC-labeled goat IgG anti-mouse IgG. Each primary mAb was used at 10 ␮g/ml. The isotype-matched control mAb (MOPC31C) is shown as the dotted line. Results are representative of a total of ﬁve separate experiments.

used to compare all treatment groups vs the control group. Values of p Յ 0.05 were considered to be signiﬁcant. Results Skin and lung mast cells express Fc␥RIIa and not Fc␥RIIb Microarray analysis with Affymetrix gene chips were used to examine gene expression in cultured human skin mast cells. As shown in Table I, expression of Fc␥RIIa was detected along with strong expression of Fc␧RI␣,Fc␧RI, and Fc␧RI, but expression was absent for Fc␥RIIb and Fc␥RIIc along with Fc␥RI, Fc␥RIII, and Fc␧RII. To conﬁrm the microarray analysis, RT-PCR with

by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd. Fc␥RII-specific primers (36, 37) was performed. RNA from cultured skin mast cells yielded RT-PCR products representative of Fc␥RIIa (Fig. 1A, upper panel), while no Fc␥RIIb products were detected in three separate cultures. An Fc␥RIIc product was detected in one of three cultures (data not shown). The identities of the Fc␥RII PCR products from positive controls and from cultured skin mast cells FIGURE 1. Fc␥RII mRNA and protein expression in human skin mast were confirmed by Southern blotting with labeled probes that recog- cells. A, RT-PCR. RNA preparations from skin mast cells and peripheral nize gene-specific internal sequences (Fig. 1A, lower panel). blood leukocytes (ϩ, positive control) were reverse-transcribed and Western blotting was used to analyze gene expression at the

http://classic.jimmunol.org ␥ ␥ ␥ Fc RIIa, Fc RIIb, and Fc RIIc transcripts were selectively ampliﬁed. protein level. As shown in Fig. 1B,Fc␥RIIb protein was detected PCR products were separated by electrophoresis on 2% agarose gels and in extracts of peripheral blood leukocytes, but not in extracts of detected by ethidium bromide staining (upper panel). Reaction mixtures with primers only (no RNA/DNA template) served as negative controls two preparations of cultured skin mast cells using Ab163. In con- ␥ (data not shown). These results were representative of three independent trast Ab260 against the cytoplasmic tail of Fc RIIa revealed a experiments, meaning that cells were obtained from three separate donors. band migrating at ϳ43 kDa from U937 cells as well as mast cells, Southern blotting performed with a digoxygenin-UTP-labeled probe rec- as expected for the glycosylated form of this receptor. To deter-

Downloaded from ognizing all Fc␥RII isoforms was used to confirm amplification of this mine whether this expression pattern reflected the conditions of gene. The results are representative of studies performed on two separate culture, Western blots also were performed with skin mast cells ␥ skin mast cell cultures. The two bands in Fc RIIC blots represent donor- that had been purified by cell sorting using anti-Fc␧RI and anti- dependent allelic polymorphisms (57). B, Western blot. Lysates from two CD117 mAbs within 36 h after their dispersal and enrichment by separate mast cell cultures (SMC1 and SMC2) of 6–8 wk were separated by SDS-PAGE under reducing conditions, and analyzed by immunoblot- Percoll density-dependent sedimentation. As shown in Fig. 1C, ting with rabbit anti-Fc␥RIIa (Ab260) (lower panel) or rabbit anti-Fc␥RIIb Fc␥RIIa, but not Fc␥RIIb, was detected in such mast cells. In (Ab163) (upper panel). As a control, the Fc␥RIIa-expressing cell line U937 contrast, Fc␥RIIb, but not Fc␥RIIa, was detected in cord blood- and Fc␥RIIb-expressing peripheral blood leukocytes (PBL) were included. derived mast cells and peripheral blood basophils as noted previ- Each lane contains 2 ϫ 105 cell equivalents. Molecular mass markers are ously (24, 36). Thus, freshly dispersed and cultured human skin indicated to the left of these gels. C, Western blot of freshly dispersed mast mast cells do not express the Fc␥RIIb isoform but do express cells. Lysates from freshly dipersed mast cells were tested as in B. These mast Fc␥RIIa. cells had been purified by sorting using FITC-labeled anti-Kit and anti-Fc␧RI mAbs within 2 days after their dispersal from skin. Peripheral blood basophils Flow cytometry was used to reveal the surface expression of ␥ ␧ (PBB) and cord blood-derived mast cells (CBMC), obtained as described (24, Fc R, Fc RI, and Kit on skin-derived human mast cells as shown 36), served as positive controls for CD32b, while Raji cells served as a positive in Fig. 2. As expected, the vast majority of cells expressed Fc␧RI control for CD32a. Each lane contains 5 ϫ 105 cell equivalents. and Kit. Surface expression of Fc␥RII was detected with IV.3 The Journal of Immunology 697

(Fc␥RIIa), AT10 (Fc␥RIIa, Fc␥RIIb, and Fc␥RIIc), and 2E1 release of 17–64% of ␤-hexosaminidase in response to 0.01 ␮g/ml (Fc␥RIIa, Fc␥RIIb, and Fc␥RIIc) (37, 42, 43), but not with 41H16 to 5 ␮g/ml of IV.3, with maximal release observed by 0.1 ␮g/ml (Fc␥RIIb and Fc␥RIIc). This pattern of labeling indicates that of IV.3 (Fig. 3A). Activation through the Fc␧RI with mAb 22E7 membrane expression of Fc␥RII isoforms is limited to Fc␥RIIa. released ␤-hexosaminidase to a similar degree, ranging from 15 to Analogous to earlier studies in which cultured human mast cells 33% in response to 0.01 ␮g/ml to 1 ␮g/ml of 22E7. Spontaneous were examined, no Fc␥RI (CD64) or Fc␥RIII (CD16) expression release of ␤-hexosaminidase was Ͻ5%. When Fc␥RIIa and was detected in resting cells (10). Fc␥␧RI were challenged simultaneously, degranulation levels were comparable with those with IV.3 stimulation (0.1–5.0 ␮g/ml) ␥ Activation of mast cells through direct Fc RIIa cross-linking alone; at the lowest dose of IV.3 (0.01 ␮g/ml) degranulation in- The functionality of Fc␥RIIa on skin mast cells was examined by creased with simultaneous Fc␧RI cross-linking, but only to the challenging them with the Fc␥RIIa-speciﬁc mAb IV.3. A dose- degree expected with Fc␧RI cross-linking alone. To ensure that dependent degranulation response was observed as measured by IV.3-mediated activation had occurred with the unaggregated by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 3. Degranulation by skin-derived mast cells after Fc␥RIIa and/or Fc␧RI cross-linking. A, Degranulation dose response. ␤-Hexosaminidase release was determined after 30 min of incubation with the mAbs as indicated. Release values were greater than the buffer control (p Ͻ 0.05) at all mAb concentrations. B, Gel filtration of IV.3 mAb. IV.3 mAb (0.5 mg/180 ␮l) was loaded onto a Superose 12 HR 10/30 column (Pharmacia) using a Shimadzu LC-10Avp HPLC system (Shimadzu) at a flow rate of 1 ml/min. The column was equilibrated and run with PBS. Elution fractions were collected at 0.5 ml/tube at 1 ml/min. The OD of each fraction was monitored at 280 nm and plotted. Gel filtration standards are shown at their elution positions for Blue Dextran (BD, ϳ2 ϫ 106 Da), thyroglobulin (Tg, 669,000 Da), and BSA (66,000 Da). C, Activation of skin mast cells with unaggregated IV.3 mAb. IV.3 in fractions from the ascending (#20), peak (#22), and descending (#24) portions of the elution profile along with prechromatography IV.3 and 22E7 were each used at 1 ␮g/ml to stimulate skin mast cells. ␤-Hexosaminidase was measured as a marker of degranulation. D, Time-course. ␤-Hexosaminidase release was determined after mast cells were incubated with 22E7 (1 ␮g/ml) and/or IV.3 (0.1 ␮g/ml) mAbs for 0, 5 and 30 s, and for 3 and 15 min. E, Degranulation of freshly dispersed skin mast cells. Mast cells were dispersed from skin, enriched to ϳ5% purity by Percoll density-dependent sedimentation and then stimulated with 22E7 (1 ␮g/ml) or IV.3 (1 ␮g/ml) mAbs for 30 min. Degranulation was assessed by measuring release of tryptase. Spontaneous p Ͻ 0.05 ,ء .release and release with a nonimmune IgG-isotype control were each Ͻ4%. Mean Ϯ SE values from three independent experiments are shown by ANOVA when experimental values are compared with 0 ␮g/ml of IV.3 within each 22E7 group in A, and to the buffer control in D and E. 698 Fc␥RIIa ON HUMAN MAST CELLS

mAb, mast cell activation was performed with IV.3 that had been subjected to gel filtration (Fig. 3, B and C). Portions of the peak fractions and those on either side along with prechromatography IV.3 and 22E7 Ab (each adjusted to a concentration of 1 ␮g/ml) were used to stimulate skin mast cells. IV.3 from the peak fraction (#22) as well as those from the ascending (#20) and descending (#24) portions of this peak induced a similar magnitude of mast cell activation when compared with the unfractionated IV.3, indicating that free (nonaggregated) IV.3 was responsible for stimu- lating these mast cells to degranulate. To compare the time courses for degranulation after Fc␥RIIa and Fc␧RI cross-linking, mast cells were stimulated with IV.3 and/or 22E7 at 1 ␮g/ml concentration for 0, 5, and 30 s, and for 3 and 15 min. As shown in Fig. 3D, the time courses of ␤-hexosaminidase release were similar in each case, with significant degranulation being detected by 30 s. Maximal release values also were similar. The functionality of Fc␥RIIa on freshly dispersed skin mast cells was examined by measuring tryptase release after stimulation with optimal amounts of anti-Fc␧RI and Fc␥RIIa mAbs. By measuring tryptase release, in contrast with ␤-hexosaminidase, the mast cell source of both the releasate and the retentate could be assured. As shown in Fig. 3E, cross-linking these receptors led to comparable tryptase release values that were significantly higher than those observed with the buffer or IgG controls, indicating that both of these receptors were functionally present on freshly dispersed skin mast cells. The release of PGD (Fig. 4A) and LTC (Fig. 4B) were as- FIGURE 4. Release of PGD (A) and LTC (B) by skin mast cells stimu- 2 4 2 4 ⌭ lated by cross-linking Fc␧RI (22E7), Fc␥RIIa (IV.3) or both receptors simul- sessed next. Similar to 22 7 stimulation, IV.3-stimulated skin 6 mast cells produced comparable levels of PGD in a dose-depen- taneously. PGD2 and LTC4 release were determined after skin mast cells (10 2 cells/ml) had been incubated with the indicated mAbs for 45 min in AIM-V dent pattern from 0.001 to 1 ␮g/ml. However, no synergistic or medium. Results from three independent experiments, each single experiment additive effect was observed when Fc␥RIIa and Fc␧RI were si- p Ͻ 0.05 by ANOVA when experi- ␧ ,ء .performed in triplicate, are presented multaneously but independently cross-linked with Fc RI. LTC4 mental values within each group are compared with the buffer control. production was not observed at any of the concentrations of 22E7 by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd.

FIGURE 5. Cytokine production by skin-de- http://classic.jimmunol.org rived mast cells challenged with anti-Fc␥RIIa and/or anti-Fc␧RI mAbs. A, IL-5. B, IL-6. C, IL-13. D, GM-CSF. E, TNF-␣. Mast cells (106 cells/ml) were incubated with the indicated mAbs for 24 h in AIM-V medium containing SBTI and SCF. Results from three independent experiments, each single experiment performed Downloaded from in triplicate, are presented as box plots (because not all data followed a normal distribution) where the hatch mark is the median and the upper and lower ends of the box are the 25th and p Ͻ 0.05 by ANOVA when ,ء .75th percentiles values for each cytokine were compared with those for the buffer control; †, p Ͻ 0.05 against IV.3 within the same mAb dose group. The Journal of Immunology 699

both IgE and IgG immune complexes. In each case, the magnitude of degranulation diminished with lesser amounts of immune complex. Coaggregation of Fc␧RI and Fc␥RIIa. The effects of coaggregating Fc␧RI and Fc␥RII using IgE and IgG anti-Der p2 mAbs were next examined. For these experiments, mast cells were sensitized with IgE to Der p2, IgG to Der p2, or both. Cells were then challenged with different concentrations of aggregated Der p2. As seen in Fig. 6B, degranulation occurred when IgE- or IgG-armed mast cells were exposed to Der p2. When mast cells were armed with both isotypes, Fc␧RI coaggregation with Fc␥RIIa led to significantly higher amounts of degranulation than to either mAb by itself ( p Ͻ 0.05), even though this increase was additive at best. Importantly, no inhibition of IgE-mediated activation was observed when IgG and IgE were coaggregated by Ag. Discussion The novel finding that human skin-derived mast cells normally express functional Fc␥RIIa, but not Fc␥RI, Fc␥RIIb, or Fc␥RIII, implies a potential role for mast cells in IgG immune complex- mediated processes in vivo. This may be analogous to the role that Fc␥RIII plays in rodent mast cells whereby mast cells become involved in IgG-mediated hypersensitivity disorders such as the Arthus reaction (7), or in IgG-mediated anaphylaxis (44). Mice, FIGURE 6. Activation of skin-derived human mast cells by Ag through unlike humans, express only the inhibitory Fc␥RIIb form of this Fc␥RIIa and Fc␧RI pathways. A, Activation by preformed IgG anti-NP–NP- receptor (45). Transient expression of Fc␥RI on human mast cells BSA and IgE anti-NP–NP-BSA immune complexes. Immune complexes con- (10) also might contribute to IgG-mediated activation of these ␮ ␮ ␮ ␮ taining 8.7 g/ml Ab with 0.13 g/ml NP-BSA, 4.4 g/ml Ab with 0.7 g/ml cells. NP-BSA, and 2.2 ␮g/ml Ab with 0.3 ␮g/ml NP-BSA were incubated with skin A current theory predicts that the allergen–IgE-mediated re- mast cells for 30 min. Supernatants and cell lysates were prepared for ␤-hexosaminidase measurements. Data are expressed as mean Ϯ SE from three sponse of mast cells will be attenuated by allergen-specific IgG independent experiments, each performed in triplicate. B, Co-crosslinking induced by allergen immunotherapy and acting by coaggregating Fc␥RIIa and Fc␧RI by Ag. Skin-derived mast cells that had been sensitized ITIM-containing Fc␥RIIb with ITAM-containing Fc␧RI receptors with Der p2-specific IgE, Der p2-specific IgG, or both mAbs were challenged (45–47). The expression of Fc␥RIIb on rodent mast cells, human with aggregated Der p2. Releasates were collected after 30 min to measure basophils, and human cord blood-derived mast cells and the ability

by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd. mediator release as described in materials and methods. Data expressed as of this receptor to inhibit Fc␧RI-dependent activation (36, 45) sup- mean Ϯ SE from three individual experiments. *, p Ͻ 0.05 by ANOVA when ports this paradigm. Indeed, the therapeutic potential for regulating experimental values are compared with the Ab-only control in A. Fc␧RI-mediated activation by coaggregation of ITIM-containing receptors led to the development of GE2, human IgG1 Hinge- CH␥2-CH␥3 region linked to the human IgE CH␧2-CH␧3-CH␧4 ␥ tested. In contrast, Fc RIIa cross-linking induced release of LTC4 region. GE2 showed dose- and time-dependent inhibition of Ag- at 0.1 and 1 ␮g/ml; this release was not signiﬁcantly altered when driven IgE-mediated histamine release from human basophils and Fc␥RIIa was simultaneously challenged with Fc␧RI. cord blood-derived mast cells and increased inhibition of IgE-me- We next examined the effects of Fc␥RIIa stimulation on the diated passive cutaneous anaphylaxis in human Fc␧RI-␣ trans-

http://classic.jimmunol.org release of cytokines (Fig. 5). Stimulation of skin mast cells by genic mice (23, 24, 48). Whether the inhibitory capabilities of GE2 cross-linking Fc␧RI and Fc␥RIIa by themselves or simultaneously act solely through the ITIM domain of Fc␥RIIb or also by com- resulted in dose-dependent increases in production of IL-5, IL-6, peting with Ag-speciﬁc IgE and/or IgG for binding to their recep- IL-13, GM-CSF, and TNF-␣ over a 24-h interval. At 0.01 ␮g/ml tors remains to be fully understood. of mAb, IV.3 resulted in signiﬁcantly less production of IL-5, GM- However, the absence of Fc␥RIIb and the presence of Fc␥RIIa CSF, and TNF-␣ than did 22E7. However, in no case did stimu- and possibly Fc␥RIIc on human skin-derived mast cells argues

Downloaded from lation with both mAbs together result in a signiﬁcantly higher level against this mechanistic explanation for the efﬁcacy of immuno-

of cytokine secretion than the sum of cytokine released by the two therapy, at least for the MCTC type of mast cell that predominates mAbs alone. Although no synergistic or additive effects were de- in skin (49). In fact, production of IgG against allergens might lead ␥ tected when cells were simultaneously stimulated through both to activation of MCTC cells through Fc RIIa. The current study Fc␧RI and Fc␥RIIa pathways, there also was no inhibition of using skin-derived mast cells found no evidence for inhibition of Fc␧RI-mediated release of these cytokines when Fc␥RIIa was si- degranulation when Fc␥RIIa and Fc␧RI were simultaneously but multaneously cross-linked. independently cross-linked with Ags or anti-receptor mAbs or when co-cross-linked with Ag. In fact, co-cross-linking led to a Activation of skin-derived mast cells by Ag higher level of degranulation than with either Fc␥RIIa or Fc␧RI Immune complexes. To test whether IgE or IgG immune com- cross-linking alone. Whether these observations can be extended to

plexes would activate skin-derived mast cells, cells were chal- MCTC cells from other tissues, or to the MCT type of mast cell that lenged with either IgG anti-NP–NP-BSA or IgE anti-NP–NP-BSA predominates in lung and small bowel mucosa remains to be stud- immune complexes. Degranulation was assessed by measuring ied. The ﬁnding of Fc␥RIIb, but neither Fc␥RIIa or Fc␥RIIc, in ␤-hexosaminidase release. The result, as shown in Fig. 6A, clearly cord blood-derived mast cells (24) distinguishes this in vitro-

demonstrated immune complex-mediated mast cell activation for derived mast cell from skin-derived MCTC cells, which may reﬂect 700 Fc␥RIIa ON HUMAN MAST CELLS

differences in the progenitors, the conditions for development, or 10. Okayama, Y., A. S. Kirshenbaum, and D. D. Metcalfe. 2000. Expression of a functional high-affinity IgG receptor, Fc␥ RI, on human mast cells: up-regulation the stage of maturation of these mast cells. ␥ J. Immunol. ␥ by IFN- . 164: 4332–4339. Fc RIIa cross-linking on the surface of skin MCTC cells leads to 11. Chong, H. J., B. L. Andrew, D. P. Bailey, H. Wright, C. Ramirez, A. Gharse, degranulation and secretion of newly generated lipids and cyto- C. Oskeritzian, H. Z. Xia, J. Zhu, W. E. Paul, et al. 2003. IL-4 selectively enhances Fc␥RIII expression and signaling on mouse mast cells. Cell Immunol. kines. Although mostly comparable to what is observed with 224: 65–73. ␧ Fc RI cross-linking, the one difference pertains to LTC4 secretion, 12. Daeron, M. 2000. Fc receptors and allergies. Rev. Fr. Allergol. 40: 445–465. which follows cross-linking of Fc␥RIIa but not Fc␧RI. The ab- 13. Daeron, M., O. Malbec, S. 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