Lymphocyte Fc receptors: the immunobiology and pathology of CD23.

Immunobiol., vol. 185, pp. 235-267 (1992)

Department of Pathology, The University of Iowa College of Medicine, Iowa City, IA 1 University of Minnesota College of Dentistry 2 Institute of Human Genetics, The University of Minnesota, Minneapolis, MN, USA

Lymphocyte Fc Receptors: The Immunobiology and Pathology of CD23 RICHARD G . LYNCH, MATYAS SANDOR, RAFAEL NUNEZ, AMBIKA MATHUR 1 , MICHAEL HAGEN, THOMAS WALDSCHMIDT, BRIAN VAN N ESS2, KEATSNELMS 2, NANCY NOBEN, ALEXANDERIBRAGHIMOV, DANA MORDUE, RANDY SACCO, PHATARAPORN TEERARATKUL, W. TIMOTHY SCHAIFF, and LEONID IAKOUBOV

I. Introduction Immunoglobulins (Ig) are cytophilic for many types of somatic cells and depending on the cell type involved, the bound Ig participates in a diversity of biological functions. This review will focus on those Ig receptors on T and B lymphoid cells that are designated as Fc receptors (FcR) because they recognize epitopes located in the constant region domains of Ig heavy chains. FcR on lymphocytes generally bind Ig monomers with low affinity (Kd= 10-7 to 10-8 mil), but the physiologically relevant binding may be high avidity interactions in which polyvalent forms of Ig-ligands cluster receptors on the surface of the lymphocyte. Other Ig-binding molecules, such as lectins and enzymes that recognize the carbohydrate moieties on Ig molecules, occur on lymphocytes, but these will not be considered in this review. During the past decade there has been a tremendous increase in knowledge of lymphocyte FcR. Based on analyses of molecular structures, patterns of independent expression, and responsiveness to lymphokines and cytokines, it is now well established that distinct receptors for each Ig heavy chain class can be expressed on the major subsets of murine lymphocytes. Lymphocyte FcR present are developmentally regulated, and once expressed they are environmentally regulated. These receptors can physically associate with MHC class II molecules and sIg on B lymphocytes (DICKLER, 1982; SCHAlfF and LYNCH, 1991), and with the CD3/TCR complex on T lymphocytes (SANDOR et aI., 1991). These characteristics along with others discussed below, suggest that FcR mediate important physiological functions on T and B lymphocytes. Some of these functions have now been identified and are discussed below and elsewhere in this volume. It is quite likely that additional functions will be described, These studies were supported in part by NIH grants CA48485 and CA49228.

236 . R. G.

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especially as more information is generated about the IgM, IgD, and IgA FcR that are expressed on the major subsets of T and B lymphocytes. Much of the new information about lymphocyte FcR will be covered elsewhere in this volume, so this review will concentrate on the results of investigations in the mouse that have been carried out in this laboratory. The emphasis here will be on the low affinity IgE FcR (CD23) that occurs on T and B lymphocytes since the molecular structures of CD23 (KIKUTANI et aI., 1986a; IKUTA et aI., 1987; LUDIN et aI., 1987; BETTLER et aI., 1989; GOLLNICK et aI., 1990) and the gene that encodes CD23 have been described (SUTER et aI., 1987; RICHARDS et aI., 1991), the mechanisms that regulate CD23 expression have been extensively investigated, and the expression of CD23 on lymphocytes is strikingly altered in a number of diseases that have in common a profound disruption of normal B cell function.

II. The expression of FcR on T and B lineage cells is highly regulated

A. B lineage lymphoid cells For each of the five major classes of FcR (Fcf!R, FcbR, FcyR, FCER and FcaR) there are discrete periods of B cell ontogeny during which each receptor is expressed (Table 1). Based on studies of normal and transformed B cells it has been established that FCf!R, FcbR, FcyR and FcaR are constitutively expressed on the surface of pre-B cells, immature B lymphocytes and virgin, mature B lymphocytes. In contrast, CD23 appears relatively late in B cell ontogeny being first detected on B lymphocytes that co-express sIgM and sIgD. While FcyR and FeaR continue to be constitutively expressed through the stage of the plasma cell, FCf!R, FcbR and FCER are not present on B lineage cells that have undergone heavy chain class switch and no longer express sIgM and sIgD. An interesting consequence of these individual developmental patterns of FcR display on B lineage cells is

Table 1. Developmental expression of FeR on B cells B cell Stage:

Pre-B (sIg-Ia-) Immature (sIgM++ sIgD-Ia+)

FeaR FcyR FCERII Fcr.tR FeaR

+ +

+ +

+

+ +

Mature (sIgM++, sIgD++, Ia+)

Switched (sIg+, Ia+)

Secretory (sIg+/-, Ia-)

+ + + + +

+ +

+

±

±

This table is based on data from studies of different B cell tumor lines using FcR probes. The reagents and methods used to visualize the FeR are described in SANDOR et al. (1990a). Resting splenic IgM++, IgD++ B cells express distinct FcRs for all five isotypes.

Lymphocyte Fc Receptors . 237

that only mature, vlrgm, sIgM\ sIgD+ B lymphocytes simultaneously express all five classes of FcR. Activation of the virgin, mature, sIgM+I sIgD+ B lymphocyte results in a transient elevation of CD23 expression at the cell surface, following which CD23 expression ceases. The very narrow developmental window during which CD23 is present, and the influence of B lymphocyte activation on CD23 suggests that CD23 mediates its function at some point in the process of B lymphocyte activation. Although CD23 is a low affinity IgE FcR and may play a role in the regulation of IgE (reviewed in CONRAD, 1990), several lines of evidence indicate that CD23 has a functional role in B cell activation. GORDON et aI. (1987) have proposed that CD23 on human B lymphocytes is an element in the regulation of cell cycle progression following B cell activation and that B lymphocytes can be triggered thru CD23. Recent studies by CONRAD (1991) and WALDSCHMIDT (1991) provide evidence that crosslinking of sIg and FCER on B lymphocytes generates an activation signal. In contrast, it is well known from the studies of PHILLIPS and PARKER (1983) that crosslinking of sIg with FcyR generates an inactivation signal and the intracellular mechanism has been investigated by RIGLEY et aI. (1989). Based on studies of human B lymphocytes, GORDON and colleagues (1989) have proposed that CD23 may be a receptor for a B cell growth factor. Additional evidence that implicates CD23 in signal transduction is the finding (SUGIE et aI., 1991) that CD23 is physically associated with the src family tyrosine kinase p59 (fyn) that is also known to be associated with CD3/TCR complex on T lymphocytes. Consistent with a dual function for CD23 as an IgE FcR and as a participant in B cell activation is the finding of two molecular isoforms of CD23 in human B lymphocytes (YOKOTA et aI., 1988). The extracellular domains and transmembrane segments of the two isoforms are identical, but they differ in the amino acid sequences of their intracytoplasmic segments. Interestingly, one isoform is IL-4 responsive while the other is not. The finding of two isoforms provides, at least in principle, a molecular basis for a dual function of CD23 since each isoform could associate with a different cytoplasmic molecule and be linked to two distinct functional outcomes.

B. T lineage lymphoid cells There is a striking difference in the levels of FcR present on the surface of resting, mature lymphocytes of the T and B lineages. All five major classes of FcR are easily detected on mature, resting, virgin B lymphocytes using assays that measure the binding of fluorochrome-tagged Ig or anti-FcR antibodies. These same assays detect little or no FcR on resting, mature T lymphocytes. In some studies sensitive immunocytoadherence (rosette) assays have detected FcR on normal, resting T lymphocytes. When activated via the CD3:TCR complex, many T lymphocytes are induced to express FcR (SANDOR et aI., 1990a, 1990b). y6: TCR + dendritic epidermal

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cells (DEC) are unusual because they display FCER and FcyR prior to being activated. It is not yet known when in the ontogeny of T lineage cells transcription of FcR genes is initiated. DM:RON et al. (1988) have shown that transcriptional activation of an FcyR gene requires nucleotide demethylation in a 5' regulatory segment of the gene. Since FcR are expressed on some murine thymocytes during a 2-3 day window late in fetal development (SANDOR and LYNCH, unpublished data), it is possible that FcR gene transcription continues at low levels in mature, resting, peripheral T lymphocytes but that surface expression of significant levels of FcR proteins is tightly linked to some element of cellular activation. An interesting aspect of FcR on T lineage cells are the patterns of display observed on different T cell subsets. The classes of FcR that have been found on murine T lymphocyte subsets are listed in Table 2. In a recent study of a panel cloned CD4 + T cells (SANDOR et al., 1990a) we found FcR on activated CD4+ITh2 cells but little or none on activated CD4+IThl cells. CD4 +ITh2 cells expressed one or more classes of FcR and some clones expressed all five classes of FcR (Fig. 1). When FcR were detected on CD4+ IThl cells their level of expression was up to 100-fold less than on CD4+/Th2 cells (Fig. 1). Interestingly, there was a distinct preference for expression of Fc[!R, FeaR and FcoR on Th2 clones. FCER and FcyR, while present on some Th2 clones at low levels, are uncommon. CD8+ T lymphocytes can also be induced to express FcR. In vivo studies of mice bearing Ig-secreting plasmacytomas and hybridomas have established that Fc[!R, FcyR, FcaR and FCER are induced on CD8+ T lymphocytes in these mice (HOOVER and LYNCH, 1980; MATHUR and LYNCH, 1986; MATHUR et al., 1986). Interestingly, the specificity matches the heavy chain class of the Ig secreted by the plasmacytoma or hybridoma. Based on a number of different observations it appears that the growth of the tumor is accompanied by the activation of host CD8+ T lymphocytes. Activation of the CD8+ T lymphocytes induces the expression of multiple classes of FcR, but only the class of FcR that matches the monoclonal Ig persists. It appears that the monoclonal Ig maintains expression of the corresponding class of FcR on the CD8+ T lymphocytes, possibly by ligand-induced upregulation of the receptor. In a preliminary study of a small number of long-passaged CD8+ CTL clones we failed to detect cell surface FcR on any of the clones (unpublished data). Since FcR are present on some in vitro lines of CD8+ T lymphomas, the lack of FcR on long-passaged CD8+ CTL clones could reflect that CTL's are a subset of CD8+ cells that do not express FcR, or that they do so only during a discrete developmental window that is not represented in the CTL clones. Alternatively, the lack of FcR on longpassaged CD8+ CTL clones could reflect the absence in vitro of a factor required for FcR expression on activated CD8+ T cells. Support for that possibility comes from the finding that when FCER+ CD8+ T cells from mice bearing IgE-secreting hybridomas are cultured in vitro, FCERs are rapidly lost (unpublished data).

240 . R. G.

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In our in vitro studies of FcR on activated T lymphocytes, activation was triggered by: a) mitogenic anti-CD3 monoclonal antibody, b) anti-TCR (idiotypic) antibody, and c) specific antigen presented by MHC-matched APC. Previous studies from other laboratories had reported FcR induction on T lymphocytes undergoing MLR (FRIDMAN and GOLSTEIN, 1974) or responding to mitogens (NEUPORT-SAUTES et al., 1975; KAWENISHI et al., 1982; ADACHI et al., 1986). Interestingly, as shown in Figure 4, when CD4+ T lymphocytes are activated via their CD3/TCR complex, FcR are preferentially induced on the Th2 subset of cells (SANDOR et aI., 1990a). The paucity of FcR expression on TCR-activated Th1 cells indicates that it is not TCR-mediated activation per se that induces FcR display. Supernatants of activated Th2 clones do not induce FcR on Th1 cells and supernatants of activated Th1 clones do not block the induction of FcR on Th2 cells.

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Figure 2. The expression of FCERII (upper left) and FcyRIII (upper right) on CD3+ Dendritic epidermal T cells (DEC). The lower panels show FCERII expression (lower left) and FcyRIII expression (lower right) on CD3+ gated DEC in the presence (clear area) and absence (shaded area) of mitogenic anti-CD3 epsilon (1452.Cll).


Moreover, when cloned Thl and Th2 cells are co-cultured and simultaneously co-activated via their CD3/TCR, FcRs are preferentially induced on the Th2 cells (SANDOR and LYNCH, unpublished data). These findings imply that soluble factors released by activated CD4+ T lymphocytes cannot account for the differential induction of FcR on Th2 and Thl cells. One possibility is that the preferential induction of FcR on CD3/TCRactivated Th2 cells reflects differences in intracellular signaling pathways in Thl and Th2 cells (GAJEWSKI et aI., 1989). Besides in vitro studies, we have examined in vivo activated T lymphocytes for FcR and have found elevated levels of FCIlR and FcaR on polyclonal CD4+ cells in mice given a single injection of mitogenic antiCD3 monoclonal antibody or a single injection of the super-antigen SEB. However, following multiple injections of anti-CD3 or the superantigen (SEB), some CD4+ T cells express a high level of FCER and/or FcyR (Fig. 3).

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Figure 3. The expression of FCERII on splenic CD4+ T cells from mice repeatedly injected with mitogenic anti-CD3 antibody (145-2Cll), upper panels, or with superantigen (staphylococcal enterotoxin B), lower panels. FCERII was detected by monoclonal antibody (B3B4), left panels, or by IgE binding, right panels.

242 . R. G. LYNCH et al.

The linkage of FcR induction to T lymphocyte activation is not limited to TCR + population of T lymphocytes. In a sequence of studies we have shown that FcR are induced on y:6 TCR+ cells activated in vitro via the CD3/TCR complex (SANDOR et aI., 1992). This has been shown for murine y:6 T lymphocytes from the spleen, intestine (lEL), and the skin (DEC). Moreover, when the anti-CD3 activation stimulus was removed, the y:6 TCR + cells progressively lose FcR expression, but when reactivated, FcR were reinduced. In vivo studies carried out in normal and y:6!TCR transgenic mice have failed to detect FcR on resting splenic, thymic, Peyer's patch, lymph node and blood y:6 TCR+ cells. Interestingly, we did find that resting dendritic epidermal y:6!TCR+ lymphocytes from murine skin (DEC) express CD23 and the CD16 form of the low affinity IgG FcR (Fig. 2). To date these are the only resting T lymphocytes in mice upon which we have detected FcR. Interestingly, activation of DEC via the CD3/TCR complex results in the loss of CD23, a slight reduction in CD16 (Fig. 2), and the appearance of FeaR and Fc!!R. Additional evidence that links FcR induction and T cell activation has come from investigations of T lymphocytes in mice infected with Schistosoma mansoni. As discussed below, the activated y:6/TCR + cells present in the granulomas of mice infected with Schistosoma mansoni express FcR (SANDOR et aI., 1992). a:~

C. Relationships between FcR and the cognate receptors for antigen on T and B lymphocytes

As more information about FcR has been developed, several interesting relationships have been identified between FcR and the cognate receptors for antigen on T and B lymphocytes. At the molecular level two interesting relationships have been found. The first is that, with the exception of CD23, all the FcR whose structures are known are encoded by members of a gene Table 2. Expression of FcRs on subsets of activated T cells T cell subset CD4+, Thl CD4+, Th2 CDS+, splenic y/o TCR+, IEL y/o TCR+, DEC'·""· y/o TCR+, DEC

FcaR

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+ + +

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This table summarizes data on normal murine T lymphocytes activated in vitro the CD3/TCR complex, except for the y/o TCR+, DEC""'" group which were freshly obtained DEC that had not been activated in vitro. " FCER were found in an occasional CD4+ ITh2 clone after in vitro activation, the vast majority of the clones examined did not bind IgE or react with B3B4 anti-CD23 monoclonal antibody (see SANDOR et aL, 1990a). ".". FcyR were found at very low levels on some CD4+ ITh2 clones after in vitro activation (see SANDOR et aI., 1990a).


superfamily whose membership includes the genes that encode the cognate antigen receptors on T and B lymphocytes, MHC molecules and a host of membrane receptors involved in immune recognition. Secondly, it has been found that FcR and the receptors for antigen on T lymphocytes can share structural subunits. For example, the zeta-chain subunit of CD3 is physically associated with the CD16-II form of the low affinity IgG FcR on human NK cells (LANIER et aI., 1989). In other studies, transfection of COS7 cells with CD16-II cDNA alone did not result in surface expression of CD16, but co-transfection with the y-subunit of the rat high affinity IgE receptor (FcERI) resulted in CD16 expression (KINET and RAVETCH, 1991). Therefore, it appears that the y-chain subunit and the zeta chain of CD3 can be used interchangeably to support expression of CD16-II, and that CD3zeta and the y-subunit of FCERI, both known signal transduction molecules, can co-associate with a number of receptors in different types of cells and can be used interchangeably. The discovery that y-chains and zeta chains can be exchanged is particularly interesting because some cells, such as certain y6 T cells, normally coexpress CD16 and CD3. Were exchange to occur between the y-chains and zeta chains in these cells, it could have novel functional consequences. Another interesting connection between FcR and TCR is their association with cytoplasmic tyrosine kinases. It was previously shown that p59 (fyn), a src-family protein tyrosine kinase, physically associates with the CD3/TCR complex. Recent studies by YODOI and colleagues have shown that p59 (fyn) can also physically associate with CD23, the low affinity IgE FcR on lymphocytes (SUGIE et aI., 1991). This finding is particularly interesting in view of the putative role of CD23 in lymphocyte activation. Additional evidence that links lymphocyte FcR with the antigen receptors on T and B lymphocytes are the many observations which show that engagement of antigen receptors on T and B lymphocytes modulates the expression of FcR. As discussed above, freshly obtained, resting T lymphocytes do not express FcR, or express them at levels below the sensitivity of conventional assays, but when activated thru the CD3/TCR complex some subsets of T lymphocytes are induced to express FcR (reviewed in LYNCH and SANDOR, 1990). On B lymphocytes, where FcR are constitutively expressed, antigen-triggered activation results in the down regulation of Fcl-tR and FCER. The modulation of FcR on T and B lymphocytes by ligation of their antigen receptors is selective since crosslinking a number of other surface membrane molecules on these cells does not modify the display of FcR. We have also found that FcR and the antigen receptors on activated T and B lymphocytes co-localize. Anti-Ig-induced aggregation and capping of sIgM or sIgD on B lymphocytes is accompanied by coaggregation, co-capping and co-shedding of Fcl-tR (ScHArFF and LYNCH, 1991). Likewise, the FcR induced on T lymphocytes by activation with mitogenic anti-CD3 monoclonal antibody have been shown to co-localize and co-migrate with the CD3/TCR complex on the surface of the T lymphocyte (SANDOR et aI., 1991). In addition to their association with the


antigen receptors on T and B lymphocytes, FcR have been shown to physically associate with MHC class II molecules on B lymphocytes (BONNEY et aI., 1988; DICKLER, 1982; SCHAIFF and LYNCH, 1991). Collectively, these findings suggest that the FcR on T and B lymphocytes participate in physiological functions related to cellular activation.

D. Modulation of lymphocyte FcR expression by environmental factors As discussed above, the presence of FcR on lymphocytes is under tight developmental control and is also strongly influenced by the state of activation of the lymphocyte. A third and major influence on lymphocyte FcR expression is the regulatory control exerted by soluble factors such as cytokines, lymphokines and Ig ligands. This subject has recently been reviewed (LYNCH and SANDOR, 1990). A general principle that appears to operate is that the soluble factors that modulate lymphocyte FcR appear to influence cells that already express FcR. The fourth major group of modulators of FcR consists of specific diseases in which alterations in FcR have been found. It is likely that as more is learned about the mechanisms that modulate lymphocyte FcR in disease, many will be found to reflect the influence of the developmental, activation or soluble factor elements of control discussed above. Table 3 lists the modulators that have been identified for B lymphocyte CD23. Several cytokines have been shown to modulate CD23 expression on B lymphocytes. Interleukin-4 (IL-4) is a strong inducer of CD23 on resting, virgin B lymphocytes in mice (CONRAD et aI., 1987) and humans (KIKUTANI, 1986b). IL-4 upregulates B lymphocyte CD23 by increasing its rate of biosynthesis (CONRAD et aI., 1987). IFN-y blocks the up regulation of

Table 3. Modulators of CD23 on B lymphocytes

Modulator

CD23 Expression

Species

References

KIKUTANI et al. (1986) CONRAD et al. (1987) Decreased BERG &LYNCH (1991) TGF-~ Increased YODOI et al. (1979) IgE LEE and CONRAD (1986) Mouse Reviewed in CONRAD (1990) LPS/a-Ig Increase (early) Decreased (late) Mouse Reviewed in CONRAD (1990) Mouse NOBEN et al. (1991) Trypanasoma brucei Decreased Mouse&Human NOBEN et al. (1991) Leishmania donovani Decreased Decreased Mouse BERG and LYNCH (1991) Plasmacytomas Decreased Human GORDON et al. (1991) anti-CDl9 Blocks IL-4 effect CONRAD et al. (1987) IFN-y Mouse Blocks IL-4 effect Human GALLIZI et al. (1988) PGE2 GORDON et al. (1990) anti-CD40 Blocks TGF-~ effect Human GORDON et al. (1990) anti-CD72 Blocks TGF-~ effect Human IL-4

Increased

Human Mouse Mouse Rat

Lymphocyte Fe Receptors . 245

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CD23 by IL-4 and alone is a weak inhibitor of CD23 expression (CONRAD et aI., 1987). IFN-a has been shown to block IL-4 induced upregulation of CD23 on human B lymphocytes (GOLIZZI et aI., 1988). We have found that transforming growth factor-beta (TGF-~) is a potent downregulator of CD23 on murine B lymphocytes (BERG and LYNCH, 1991). The molecular mechanism by which TGF-~ downregulates B lymphocyte CD23 is unknown. TGF-~ blocks the IL-4 induced upregulation of CD23 but not the IL-4 induced upregulation of sIgM and class II MHC on B lymphocytes (BERG and LYNCH, 1991) (Fig. 4). GORDON et ai. (1991) have reported that monoclonal antibodies directed to CD19, CD40 and CD72 influence CD23 expression on human B lymphocytes. Many laboratories have reported that immunoglobulins can modulate FcR expression. Each heavy chain class of Ig has been shown to upregulate the expression of its FcR on lymphocytes. This has been shown for all the classes of FcR on T lymphocytes and for those classes of FcR on B lymphocytes that have been investigated so far. The mechanisms involved in Igligand induced FcR up regulation include: a) increased rate of receptor biosynthesis (YODOI et aI., 1979; HOOVER et aI., 1981; YODOI et aI., 1982); b) avidity maturation of the receptor (SANDOR et aI., 1990b); and decreased rate of receptor turnover (LEE et aI., 1987). In some studies evidence has been presented that more than a single mechanism may simultaneously operate (SANDOR et aI., 1990b). Forms of Ig-ligands that crosslink FcRs, such as antigen:antibody complexes, polymeric Ig, cell surface Ig, solid phase Ig and aggregated Ig, have been shown to modulate lymphocyte FcR. While FcR crosslinking can result in downstream consequences that alter lymphocyte effector or regulatory functions, it also increases the efficiency of ligand capture since more Ig is bound per lymphocyte at lower concentrations of Ig.

246 . R. G.

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et al.

III. The specificity of lymphocyte Fc receptors A number of laboratories have been interested in the fine specificity of Ig binding to lymphocyte FcR. A sequence of studies have sought to identify the molecular structural elements of CD23 that are required for the binding of IgE, while other investigations have addressed the structural elements of IgE that are required for binding to CD23. It has been shown that the binding of IgE to CD23 does not depend on the oligosaccharide moieties present on the epsilon heavy chain (VERCELLI et aI., 1989). This is an important finding because CD23, while not a lectin, has an ectodomain with structural homology to a family of lectins (IKUTA et aI., 1987). Moreover, BETTLER et ai. (1989a) prepared deletion mutants of human FCERII and showed that the site necessary for IgE binding is the 123 amino acid segment that shows structural homology to the carbohydrate binding domains of C-type animallectins. These studies established that the lectin homology segment of CD23 can bind a protein ligand, and also showed that the cysteine residues in this region of CD23 are required for binding IgE. Studies with monoclonal anti-IgE antibodies indicated that the CE3 domain was the site of contact with CD23 (CHRETIEN et aI., 1988; CONRAD, 1990). Experiments using recombinant and chimeric IgE molecules confirmed the CE3 domain requirement and also identified the need for CE3 domain dimers in order for IgE to bind to CD23 (VERCELLI et aI., 1989). While the main focus of the present review is on lymphocyte CD23, there are some aspects of the specificity of binding of the other classes of Ig to the other classes of FcR that differ significantly and will briefly be considered now. The binding of IgA by murine lymphocytes has been difficult to dissect for several reasons. Probably the major limitation has been the lack of precise information about the actual ligand. IgA occurs as monomers, dimers, and various sizes of oligomers. This complexity often makes it difficult in binding studies to determine which species of IgA in the mixture is actually bound to the receptor. Without that information, it is not possible to determine binding constants or quantitate receptor copy number. We have found that murine plasmacytomas differ in the amount of monomeric, dimeric and oligomeric IgA they secrete. This is an important consideration in studies that attempt to address the specificity by competitive binding assays. In our studies of murine lymphocyte IgA binding, we have found that the relevant IgA ligand is in the oligomeric (greater-thandimer) fraction of the purified IgA (WALDSCHMIDT and LYNCH, unpublished observations). Free monomeric IgA is clearly recognized by the receptor, but the affinity of binding is so low that essentially none of the monomeric IgA is retained after the cells have been washed. While dimeric IgA also binds to the FcaR on lymphocytes, the binding is weak. The selection of the IgA used in competitive inhibition assays is critical. Some preparations of IgA may contain little or no oligomeric IgA and are poor inhibitors, even when large excesses of the IgA are used. Likewise, IgA preparations that contain large amounts of oligomeric IgA are good com-

Lymphocyte Fc Receptors· 247

petitive inhibitors. The binding of IgA by murine lymphocytes is further complicated because these cells have been reported to express surface lectins and transferases that can bind IgA via the carbohydrate moieties of the alpha heavy chain (AICHER et aI., 1991). However, under the conditions we have used, the carbohydrate moieties on a-heavy chains do not appear to influence binding of IgA to the murine lymphoid cell FeaR since enzymatic removal does not alter the binding of IgA (IBRAGHIMOV, SANDOR and LYNCH, unpublished data). The murine lymphocyte FeaR does not appear to be linked to the surface membrane via a phosphoinositolglycan bridge since the receptor is not sensitive to phospholipase C (IBRAGHIMOV, SANDOR and LYNCH, unpublished data). The relationship of the murine T lymphoid cell FcaR to the recently cloned human myeloid cell FcaR (MALISZEWSKI et aI., 1990) is unclear. Using low stringency conditions, mRNA from cloned murine T lymphoid cells that bind IgA, and a cDNA for the human myeloid cell FcaR, we did not find any evidence for structural relatedness. Furthermore, we did not detect binding of polyclonal antibodies specific for the human myeloid cell FcaR to murine lymphoid cells that express high levels of FcaR (M. HAGEN, M. SANDOR, D. SPINELLA and R. G. LYNCH, unpublished observations). Using domain deletional mutants of murine IgM and Cft domain-specific monoclonal antibodies, we previously showed that the Cft3 domain was critical for the binding of IgM to normal T and B lymphocytes (MATHUR et aI., 1988a, 1988b). In unpublished studies (A. IBRAGHIMOV, M. SANDOR and R. G. LYNCH, manuscript in preparation) analyses of the fine specificity of IgM binding to a panel of T and B lymphoid cell clones suggests that more than a single form of FcftR occurs on murine lymphocytes. The results of those studies strongly suggest that conformational changes induced in the IgM molecules by anti-Cft domain antibodies profoundly influence the binding of IgM to the FcftR on some lymphoid cell subsets. As was also found for IgA, enzymatic removal of the carbohydrate from ft-heavy chains did not alter the binding of IgM by murine T and B lymphoid cells, and treatment of FcftR+ lymphoid cells with phospholipase C did not alter IgM binding (A. IBRAGHIMOV, M. SANDOR and R. G. LYNCH, unpublished observations). The binding of IgD to the Fc6R on lymphocytes is an example where the carbohydrate on Ig heavy chains appears to be critical for binding to the receptor (AMIN et aI., 1990). The fine specificity of IgG binding by FcyRs has been discussed elsewhere (BURTON, 1985).

IV. The molecular structure of lymphocyte CD23 Murine B lymphocyte CD23 is a 47 kD, single chain, type-II, membrane glycoprotein whose 332 residue amino acid sequence has been deduced

248 . R. G.

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et al.

from the nucleotide sequence of cloned cDNA isolated from B lymphoid cells (BETILER et aI., 1989a; GOLLNICK et aI., 1990). Murine B lymphocyte CD23 contains one N-linked high mannose oligosaccharide (KEEGAN and CONRAD, 1987) and is a labile receptor in that a soluble form of CD23 (sCD23) is released from the lymphocytes (RAO et aI., 1987). While there has been good agreement in the literature about the occurrence of CD23 on rodent and human B lymphocytes, there has been some disagreement about the occurrence of CD23 on T lymphocytes. Since the original report by YODOI and ISHIZAKA (1979) that rodent and human T lymphocytes express FCER, a number of laboratories have reported finding FCER on T lymphocytes (HOOVER et aI., 1981; THOMPSON et aI., 1983; SARYAN et aI., 1983; NAGAI et aI., 1985; MATHUR et aI., 1986; DELESPESSE et aI., 1986; NUTMAN et aI., 1986; PRINZ et aI., 1987; ARMITAGE et aI., 1989), while other laboratories have not found FCER on T lymphocytes (KIKUTANI et aI., 1986; DEFRANCE, 1987; RAo et aI., 1987). The results of studies performed on freshly obtained T lymphocytes from normal donors were difficult to interpret because of the small proportion of cells which appeared to express IgE receptors. Furthermore, discrepancies between published results were difficult to evaluate because different assays and reagents were often used in different laboratories. In retrospect, some of the differences reported may reflect that some laboratories were examining normal, resting T lymphocytes, while some laboratories were examining T lymphocytes from donors with diseases in which serum levels of IgE antibodies were markedly elevated. As new knowledge about the regulation of lymphocyte FcRs was developed, it provided insight into some of the early discrepancies. For example, it was subsequently shown that FCER are preferentially expressed on certain subsets of T lymphocytes (SANDOR et aI., 1990; 1991 b), that activation via the CD3/TCR complex induces FCER expression on some T lymphocytes (SANDOR et aI., 1990; 1991a), and that IgE up regulates FCER expression (MATHUR et aI., 1986; LEE et aI., 1987; LYNCH et aI., 1988). Compelling evidence that human T lineage cells could produce FCER was the finding that HTLV-1-transformed CD23+ T cells reacted with antiCD23 mAbs and contained CD23 mRNA by Northern analysis (NUTMAN et aI., 1987) and by PCR (NUNEZ et aI., manuscript in preparation). The first strong evidence that murine T cells could express FCER in vivo was our finding (MATHUR et aI., 1986) that mice bearing B53, an IgE-secreting hybridoma, had large numbers of CD8+ T lymphocytes that expressed IgEspecific receptors. Using antibodies developed by CONRAD and colleagues (RAO et aI., 1987) by immunization with B cell CD23, a 49 kD, IgE-binding protein was immunoprecipitated from the surface membranes of T lymphocytes isolated from mice bearing an IgE-secreting hybridoma (MATHUR et aI., 1988c). This was the first direct biochemical evidence that murine T lymphocytes could express IgE Fc receptors. The CD23 isolates from the surface membranes of murine Band T cells were shown to have common IgE-binding properties and molecular weights, but the T cell isolate reacted with only 1 of the 3 antibodies that reacted with the B cell isolate. These


findings suggested that CD23 on murine T and B lymphocytes have molecular structures that are very similar, but not identical. However, our in vivo studies in IgE-hybridoma mice did not formally rule out the possibility that CD23 had adsorbed to the T lymphocytes and had actually been synthesized by other cells, such as B lymphocytes. To address that issue we have examined a panel of cloned and culture-adapted CD4+ and CD8+ murine T cell lymphomas, antigen-specific T cell clones, and B lymphoma clones for the expression of CD23 mRNA. The T lymphoma panel contained representatives of various stages of T cell development and subset differentiation. From the published nucleotide sequence of murine B lymphocyte CD23 (BETTLER et aI., 1989a; GOLLNICK et aI., 1990) we synthesized eight CD23-specific oligonucleotide primers and screened the reverse transcriptase-PCR products of polyA-RNA and total cytoplasmic RNA using as a probe the cloned murine CD23 cDNA (GOLLNICK et aI., 1990). As might be anticipated from the very low level of FceR detected on murine T lymphocytes, there appears to be considerably less CD23 template in T cell RNA compared to B cell RNA. Following a first round of 30 cycles of PCR, the predicted PCR product from B cells and one T cell clone was detected on ethidium bromide minigels, but the PCR products of other T cell clones were detected only by Southern blot. When new internal primers and a second round of nested PCR were used, we found that all the T cell samples yielded CD23-related products that could be detected on ethidium bromide minigels. Some T cell populations contained a single product of the predicted size, but some T cell populations contained 3 distinct PCR products. In the studies of murine CD23 structure by GOLLNICK et aI. (1990), no evidence was found for CD23 transcripts in one murine T cell clone using a single round of PCR and ethidium bromide gel analysis, or in four T cell clones using an RNAse protection assay. Our investigations differed from the studies of GOLLNICK et al. (1990) in the conditions and primers used for PCR, and in the T cell populations analyzed. The relative ease with which investigators have detected CD23 transcripts in B cells compared to T cells may reflect that: a) resting, mature B lymphocytes constitutively express CD23 and that it is easily upregulated by IL-4, while b) resting mature T cells do not constitutively express CD23 and, even when activated via the CD3:TCR complex, only a minority of T cells are induced to transiently express CD23. In our survey of over thirty murine T cell populations, only two clones, D8 and CDC35, expressed significant amounts of IgE binding when activated via the CD3:TCR complex (SANDOR et aI., 1990a). When we examined the clone CDC35 for CD23 transcripts by PCR, a CD23-related product was detected by Southern analysis and by ethidium bromide gel, after a first round of 30 cycles (R. NUNEZ et aI., 1992). DNA sequence analysis of the PCR product established its relationship to CD23. Overall, these findings suggest that when CD23 transcripts are produced by murine lymphocytes, the amount produced by T lymphocytes is considerably less than that produced by B

250 . R. G.

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lymphocytes. It could be that some of the differences observed in CD23 expression between Band T lymphocytes reflects that the optimal conditions for CD23 induction on T lymphocytes are yet to be determined. In our recent studies, when the PCR products that hybridized with the CD23 probe under conditions of high stringency were subjected to DNA sequence analysis, we found that 6 of the 7 T cell populations contained CD23-related PCR products. The DNA sequence analyses carried out to date have shown that some of the CD23-related PCR products from T and B cells differ from the published sequence of B cell CD23 (NUNEZ and LYNCH, unpublished data). The nucleotide sequence differences are located almost exclusively in the intracytoplasmic and transmembrane segments of CD23. These findings indicate that murine CD23 exists as multiple isoforms. Of particular interest is the finding that some T and B cell clones contain a transcript that ~pcodes the extracellular domains and a small segment of the NH2-terminal portion of the cytoplasmic domain, but is devoid of the transmembrane segment and most of the cytoplasmic domain, findings that suggest a secreted form of CD23 (NUNEZ and LYNCH, 1992). A recent publication (RICHARDS et aI., 1991) presented the genomic structure of murine CD23 and showed that, similar to what had been previously found for the human gene, the murine CD23 gene spans 12.9 kb and includes 12 exons surrounded by 11 introns. The authors presented evidence that by utilization of different transcriptional initiation sites and alternative RNA splicing, more than a single CD23 mRNA can be generated from the gene. V. Functions of lymphocyte Fc receptors The list of established and putative functions of lymphocyte FcRs has grown considerably since the initial findings that antibody-mediated feedback inhibition resulted from the binding of antigen-antibody complexes by B lymphocytes (reviewed in UHR and MOLLER, 1968), and that the Fc portion of IgG was required for inhibition to occur (SINCLAIR, 1969). Recent reviews (LYNCH et aI., 1990a, 1990b) have considered the functions of lymphocyte FcRs, and the focus here will be on the functions of CD23 on lymphocytes. Since CD23 is also present on macrophages, platelets, eosinophils, thymic epithelium and other cell types, and because CD23 has been proposed to mediate a number of distinct functions, an issue frequently raised is: «How can the same molecule mediate such a diversity of actions?» It is probably relevant to this point that two isoforms of CD23, that differ only in their intracytoplasmic domains, have already been identified in humans (YOKOTA et aI., 1988), and that our recent findings suggest that isoforms of CD23 also occur in mice (NUNEZ, HAGEN, SANDOR and LYNCH, unpublished data). Additional studies are needed to determine to what extent CD23 is molecularly heterogeneous. The issue of a multiplicity of functions for a single receptor is not a new theme in the area of FcRs. The IgG FcRs


on trophoblasts and neonatal intestinal epithelium mediate immunoglobulin transport, on macrophages and neutrophilic granulocytes they mediate phagocytosis, on NK, LAK and y:b/DEC cells they mediate cytotoxic reactions, on B lymphocytes they transduce inactivation signals, and on vascular endothelial cells they trigger prostaglandin E2 release (SANDOR et aI., 1989). More information is needed to sort out the details, but it is already well established that: a) CD23 occurs on T and B lymphocytes where its expression is highly regulated and linked to cellular activation, and b) CD23 is an element in several important functional activities mediated by T and B lymphocytes. What follows is a summary of the present status of knowledge of these CD23-related activities.

A. CD23 and the regulation of lymphocyte growth and differentiation An increasing number of observations support the proposal of GORDON and colleagues (GORDON et aI., 1986; GORDON et aI., 1987; GUY and GORDON, 1987) that CD23 is involved in some aspect of B lymphocyte activation. These include the observation that IL-4, a potent growth factor for B lymphocytes, is also a strong upregulator of CD23 on B lymphocytes (KIKUTANI et aI., 1986b). Recently, GORDON et ai. (1991) have pointed out the intimate functional relationship between B lymphocyte CD23, IL-4R and CD72, the latter of which forms a receptor:ligand pair with CDS (VAN DE VELDE et aI., 1991). It has been suggested that soluble CD23 influences lymphocyte growth and development via a cytokine-like activity (GORDON et aI., 1987). WALDSCHMIDT and TYGRETT (1991) have recently shown that crosslinking CD23 to sIgD on virgin B lymphocytes induces a striking proliferation of the B lymphocytes. In a recent study, Luo et ai. (1991) reported that crosslinking of CD23 with complexes of IgE, or with antiCD23 antibody, prevented the proliferation and differentiation of B lymphocytes that is induced by anti-IgM plus IL-4 or by SAC and resulted in cell cycle arrest late in G 1. VAN LIER et ai. (1987) have reported that in a family of anti-CD3 isotype switch-variant monoclonal antibodies that included yl, y2b, y2a, E and a, only the IgE variant anti-CD3 antibody induced excellent T cell help for IgM production. While the mechanism of the effect is unknown, the finding is interesting because mature, virgin, IgM -producing B lymphocytes express all five major classes of FcR and, in principle, any of the switch variant anti-CD3 antibodies could have simultaneously engaged the CD3 on T lymphocytes and an FcR, either the constitutively expressed FcRs on B lymphocytes or the activation-induced FcRs on T lymphocytes. The finding that only the IgE variant induced help suggests a role for CD23. While the mechanism remains unknown, the finding that only those antiCD3 antibodies that could engage CD23 induced T cell help for IgM production suggests that CD23 can influence the activation process of mature, virgin B lymphocytes.

252 . R. G.

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et at.

B. CD23 and the regulation of immunoglobulin heavy-chain isotype The concept of an Ig-isotype network in which Fc receptors function as regulatory molecules was discussed by DAERON and FRIDMAN (1985) and the role of FcR+ T c ells in Ig-isotype-specific regulation was recently

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reviewed (LYNCH and SANDOR, 1990). During the past decade there has been a strong interest in the regulation of IgE production and particularly in the possibility that lymphocyte IgE FcRs and the soluble IgE-binding factors derived from them might play important functional roles. This area was covered in a recent review by CONRAD (1990). Our investigations have focused on CD8+ :FCER+ T cells and their influence on IgE production by murine hybridoma cells in vivo and in vitro. MATHUR et ai. (1986a) found that mice bearing B53, an IgE-secreting anti-TNP hybridoma, develop large numbers of CD8+ T cells that display FCER. The development of CD8+ :FCER+ T cells was followed by progressive decrease in IgE production by the B53 hybridoma cells (MATHUR et aI., 1987). The loss of IgE production was shown to be due to a specific decrease in E-heavy chain gene expression as determined by Northern blot and nuclear run-on analyses (MATHUR et aI., 1987). The decrease in E-gene expression was selective since there was no effect on kappa light chain gene expression, and the hybridoma cells continued to proliferate, leading to demise of the host. In subsequent studies, we established that the decrease in E-heavy chain expression was due to impaired transcription of the epsilon heavy chain gene (MATHUR et aI., 1990; NELMS et aI., 1991). In vivo depletion of host lymphoid cell subsets with infused monoclonal antibodies established that the inhibition of E-gene transcription was dependent on CD8+ T lymphocytes (MATHUR et aI., 1987). In subsequent studies we confirmed these results with SE1.3, an IgE-arsonate hybridoma, but not with A3B1, another IgE anti-TNP hybridoma. Mice bearing A3B1 neither developed CD8+:FcER+ T cells nor showed inhibition of IgE production by the hybridoma cells (MATHUR et aI., 1990). When a long-passaged in vitro clone of B53 was co-cultured with unfractionated spleen cells from mice with B53 hybridomas, E-heavy chain gene transcription was inhibited in the hybridoma cells (MATHUR et aI., 1990). Subset depletion and reconstitution analyses showed that inhibition of E-gene transcription was dependent on CD8+:FcER+ T cells and activated macrophages. Interestingly, when A3B1 cells were co-cultured with

Figure 5. Representative flow cytometry data of cytoplasmic E, x and CAT expression in B53 cells. B53 cells or cells recovered from co-culture were washed, stained with FITC: goat antimouse E, x, or rabbit anti-CAT protein antibodies as previously described (MATHUR et aI., 1987), and examined for cytoplasmic fluorescence using flow cytometry with a FACSean machine (MATHUR et aI., 1987). (A) Untransfected B53 cells. (B) B53 cells transfected with the Ig heavy-chain containing CAT expression plasmid (B53~7oo), (C) ~700-transfected B53 cells co-cultured for 48 h at 2:1 spleen cell:B53 cell ratios with spleen cells from normal mice [(SC)N]. (D) ~700-transfected B53 cells co-cultured for 48 h at 2: 1 spleen cell:B53 cell ratios with spleen cells from mice with B53 tumors [(SC)B53]. Cells were transfected (10 [tg plasmid/ 2 x 10 7 cells) using the DEAE-dextran protocol as described (NELMS et aI., 1990). These results were reproducible in four independent experiments. The X axis represents the relative fluorescence intensity. Background staining (shown by dotted curve) was assessed by analyzing cell samples treated with control, non-specific Ab conjugated to FITC. Peaks to the right of the vertical dotted line represent cells positively stained with the indicated FITC-conjugated Ab.

254 . R. G.

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et al.

spleen cells from mice with B53 hybridomas, E-gene transcription III the A3Bl cells was inhibited indicating that A3Bl, while defective in inducing CD8+:FcER+ T cells in vivo, was responsive to the regulatory effectors induced by the B53 hybridoma cells.

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In other studies, NELMS et al. (1991) transfected B53 hybridoma cells with f,l700 or x473, plasmids that contain the Ig-heavy and kappa-light chain intron enhancers, respectively, upstream to a c-fos promoter linked to the reporter gene, chloramphenicol acetyltransferase (CAT). When cocultured with spleen cells from B53-bearing mice, but not from normal mice, transcription of both the E-heavy chain gene and CAT gene were inhibited in the f,l700-transfected B53 cells, while transcription of the Eheavy chain gene, but not the CAT gene, was inhibited in the x473transfected B53 cells. Transcription of the kappa light chain gene remained unaltered in B53 transfectents co-cultured with spleen cells from normal or B53-bearing mice. The loss of CAT expression was detected by CAT enzymatic assays and by cytoplasmic staining for immunoreactive CAT protein (Figs. 5 and 6). These studies have identified a new regulatory mechanism dependent on CDS+:FcER+ T cells that specifically represses the Ig-heavy chain intronic enhancer, resulting in the suppression of Eheavy chain gene transcription. It is noteworthy that the spleens from B53 tumor-bearing mice also suppress IgE expression in two other IgE-producing hybridomas suggesting that this effect is isotype-specific and not idiotype-specific. This new regulatory pathway is the first indication that the enhancer mediated expression of Ig genes in B cells can be modulated through T cell dependent processes. In summary, the studies we have reviewed here have focused on the downregulation of IgE antibody expression and the role of CDS+ :FcER+ T cells in that process. However, other studies from a number of laboratories have described experimental systems that link other classes of lymphocyte FcR to the regulation of antibody production. The literature on B lymphocyte CD23 and its relationship to the regulation of IgE antibody expression has been reviewed by CONRAD (1990). FRIDMAN and SAUTES (1990) have reviewed the role of T lymphocyte FcyR and sIgGBF in the downregulation of IgG antibody expression. The participation of T lymphocyte FcaR+ T lymphocytes in the positive and negative regulation of IgA antibody production was reviewed by LYNCH and SANDOR (1990). C. L ympbocyte Fc receptors as accessory adbesion molecules

In principle, Fc receptors on lymphocytes could function as accessory adhesion molecules and facilitate cell-cell interaction. For example, the Figure 6. Representative flow cytometry data of cytoplasmic E, x, and rabbit anti-CAT expression in x473- or pRSV-CAT-transfected B53 cells. B53 cells recovered from co-culture were washed, stained with FITC: goat anti-mouse E, x, or CAT protein antibodies as previously described (MATHUR et aI., 1987), and examined for cytoplasmic fluorescence using flow cytometry (MATHUR et aI., 1987). B53 cells transfected with CAT expression plasmids containing the Ig kappa light-chain (B53 x473 ) or RSV (B53 RSV ) enhancers were co-cultured for 48 h at 2: 1 spleen cell:B53 cell ratios. (A) B53 x473 co-cultured with spleen cells from normal mice [(SC)NJ. (B) B53 x473 co-cultured with spleen cells from mice with established B53 tumors [(SC).S3J. (C) B53 RSV co-cultured with (SClN- (D) B53 RSV co-cultured with (SC)B53' These results were reproducible in four independent experiments. Data are presented as in Figure 5.

256 . R. G.

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et al.

multiple copies of Fc receptors on regulatory T lymphocytes could bind to the multiple copies of sIg on B lymphocytes in a zipper-like fashion and thereby promote cytoadhesion. The Fc receptors on T lymphocytes are functionally polyvalent as are the monomeric sIg molecules on B lymphocytes, because as membrane-anchored molecules, they both take on the polyvalent properties of solid-phase ligands. Although individual Fc receptor molecules on T lymphocytes exhibit low affinity binding of monomeric immunoglobulin ligands in solution, their functional polyvalency would result in high-avidity binding when they engaged the sIg on B lymphocytes. Moreover, the discrepancy in binding strengths between soluble and cell surface immunoglobulin would assure that serum immunoglobulin would not effectively block the cellular adhesion function. Since previous studies with domain-specific monoclonal antibodies established that all of the constant region domains of sIgM on B lymphocytes are accessible for engagement by monoclonal antibodies, the sIg on B lymphocytes should be accessible to the FcR on T lymphocytes. The possibility of an adhesion function for T lymphocyte Fc receptors could be relevant to the findings of SANDOR et al. (1990a) that CD4+/Th2 cells, when activated thru the CD3/TCR complex, are induced to express multiple classes of FcR. Since B lymphocytes are the predominant targets of lymphokine-mediated Th2 help, the display of FcR on Th2 cells following activation by specific antigen could facilitate the adhesion of Th2 cells to B cells. It is known from electron micrographs that collaborating T and B lymphocytes form intimate zones of surface membrane apposition in which a number of complementary adhesion molecules have been shown to playa central role. Our findings (SANDOR et aI., 1990; TEERARATKUL and LYNCH, 1991) suggest that FcR:sIg should be added to the list of complementary adhesion molecules involved in T:B interaction. Conceivably, the docking of B cell sIg in the FcR of the Th2 cell might trigger the release or transfer of lymphokines, or transduce a positive or negative regulatory signal. Moreover, the display of multiple classes of FcR on Th2 cells provides a mechanism for the T cell to recognize the classes of sIg being expressed by the conjugate partner B lymphocyte. It is possible that the signaling events in the Th2 cell might differ if in one case only its FC[,lR were engaged, while in another case its FcoR and FC[,lR were engaged. A role for T lymphocyte FcRs in T:B interaction is suggested in recent studies in which FC[,lR and sIgM were shown to contribute to cognate cytoconjugate formation between Th2 cells and sIgM+ B cells (TEERARATKUL and LYNCH, 1991). The constitutive expression of Fc receptors on B lymphocytes also provides a possible mechanism for B cell-B cell adhesion. For example, in the presence of IgG-immune complexes it is possible that cytoconjugates could form between FcyR+ polyclonal B lymphocytes. In principle, immune complexes could also facilitate adhesion between lymphocytes and other cells that express FcR, such as endothelium, virus-infected epithelium, macrophages, basophils, mast cells, eosinophils, platelets,


trophoblasts, NK cells, and neutrophilic granulocytes. In certain pathologic states the targets might be expanded to include bacteria, parasites and cancer cells that express FcR.

D. Lymphocyte CD23 and IgE-mediated immune responses Throughout the development of knowledge about lymphocyte CD23 there has been a tendency for investigators to segregate the possible role of CD23 in the regulation of IgE expression from its possible role in B lymphocyte growth and differentiation. However, the discoveries that CD23 could be expressed on other lymphocytes such as CD4 +ITh2 cells (SANDOR et al., 1990), CD8+ cells (MATHUR et al., 1986), and CD4-8-/y() T cells (SANDOR et al., 1991 b), identified additional regulatory and effector functions whose activities were potentially influenced by IgE. With the realization that CD23 also occurred on cells other than B lymphocytes, it was no longer simply a matter of dual roles for CD23, and it became clear that CD23 connected IgE to a multiplicity of other cells and to their specialized functions. In the discussion that follows two examples of the connection of IgE to the specialized functions of cells that express CD23 will be considered. Some of the discussion is based on experimental data and some is speculative. The first example considers a hypothetical mechanism whereby IgE, via B lymphocyte CD23, alters B lymphocyte function and modulates the potential for systemic anaphylaxis. The second example considers a hypothetical mechanism whereby IgE, via CD23 on skin y()- T cells, triggers a cytotoxic reaction that has some of the properties of a memory DTH response but which is non-MHC restricted. This «IgE-dependent» DTHlike response could be a novel element of first line defense in the skin. 1. Polyclonal B cell activation via IgE and CD23. The presence of FcRs on B lymphocytes provides a potential mechanism to amplify antigen processing and presentation. It is known that antigen bound by the endogenous sIg on normal B lymphocytes can be internalized, processed, and reexpressed on the cell surface in association with MHC class II molecules, where it can induce the activation of antigen-specific T lymphocytes. If antigen bound to antibody in the Fc receptors on normal B lymphocytes was handled in a similar fashion, then it is conceivable that at the point in antibody responses when antigen-antibody complexes are formed, a marked amplification of antigen processing and presentation would take place. To date, the only class of FcR on B lymphocytes that has been shown to participate in antigen processing and presentation is, surprisingly, the low affinity IgE FcR (CD23). This has been shown for murine (KEHRY and YAMISHITA, 1989) and human (PIRRON et al., 1990) B lymphocytes. In an elegant study, KEHRY and YAMASHITA (1989) have demonstrated that CD23 on normal murine B lymphocytes can bind IgE:antigen complexes and mediate antigen processing and presentation with an efficiency equal to that

258 . R. G.

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of B lymphocytes that initiate antigen processing and presentation via their endogenous sIg. In contrast, normal B lymphocytes fail to process and present antigen complexed to IgG antibodies and bound to the lymphocyte via their IgG FcR (KEHRY and YAMISHITA, 1989; SNIDER and SEGAL, 1989). It is interesting to speculate about the possible in vivo consequences of the observation of KEHRY and YAMASHITA. Like IgA antibodies, IgE is an antibody that is predominantly expressed at environmental barriers, such as the mucosae of the intestinal and respiratory tracts and the skin. Most of what is known about IgE function comes from studies of pathological conditions. It is generally accepted that a major function of IgE is in host defense against parasites at the site of infection, such as the intestine. A potential danger to the host is systemic dissemination of the IgE antibodies, occupancy of the high affinity FCERs on basophils and mast cells, receptor crosslinking by antigen, and systemic anaphylaxis. An interesting feature of many parasite infections is the concomitant polyclonal hypergammaglobulinemia. The basis for this polyclonal B cell activation is unknown, but one possibility is that IgE-antigen complexes bind to CD23 on host B lymphocytes and promote cellular activation. Since CD23 is involved in B lymphocyte activation, the binding of the immune complexes could directly facilitate or trigger polyclonal activation. Alternatively, lymphokines might be delivered to the polyclonal B lymphocytes by parasite-specific Th2 cells involved in cognate recognition of the parasite antigen that was processed via CD23 and presented via MHC class II by the polyclonal B cells. In either case, polyclonal B cell activation would ensue, polyclonal hypergammaglobulinemia would be seen, and if severe and protracted the host might exhibit a B cell immunodeficiency syndrome. A consequence of this speculated mechanism is that some of the polyclonally-activated B lymphocytes would actually be parasite-specific, and their activation would lead to the secretion of IgM and IgG anti-parasite antibodies. Unlike the IgE antibody:antigen complexes, the IgG antibody:antigen complexes do not amplify antigen processing and presentation via FcR on B lymphocytes (KEHRY and YAMISHITA, 1989; SNIDER and SEGAL, 1989). The polyclonal parasite-specific IgG antibodies, being present at much higher concentrations than the parasite-specific IgE antibodies, would easily compete with IgE antibodies for parasite antigen and thereby antagonize the conditions that lead to anaphylaxis. The polyclonal parasite-specific IgG antibodies induced by this proposed mechanism could be considered the naturally occurring counterparts of the so-called «blocking antibodies» of the clinical allergist. There are several mechanisms by which these antibodies could antagonize the development of systemic anaphylaxis. 2. The linkage of IgB antibodies, CD23 and y6:T cells to a form of contact hypersensitivity. As mentioned above, yo:TCR+ dendritic epidermal cells (DEC) freshly isolated from murine skin express FCER (CD23) and FcyR (CD16) (SANDOR et aI., 1991). The finding of CD23 on DEC could possibly explain a paradoxical observation previously made in TIGELAAR'S labora-


tory (R. TIGELAAR, unpublished data). In that study, normal mice infused with a monoclonal IgE anti-DNP antibody exhibited a contact hypersensitivity to dinitrofluorobenzene (DNFB) that had the characteristics of a T cell mediated, delayed-type response, but was clearly induced by the infused monoclonal IgE. It was observed that mice infused with the monoclonal IgE anti-DNP antibody responded to an initial skin challenge with DNFB as if they had previously been contact sensitized to DNFB. Athymic (nude) mice infused with the monoclonal IgE anti-DNP antibody did not exhibit the contact hypersensitivity, a finding that implied the delayed reaction was T cell dependent. However, when the same experiment was done in normal mice that had been depleted of CD4 + and CDS+ cells by infusion with monoclonal anti-CD4 and anti-CDS antibodies, the IgE-mediated hypersensitivity reaction was still observed, a finding, which at that time, implied that T lymphocytes were not involved in the delayed reaction (R. TIGELAAR, personal communication). The IgE-dependent hypersensitivity reaction was shown to be independent of mast cells in experiments that were conducted in mast cell-deficient mice. The discovery that DEC express CD23 and bind IgE (SANDOR et aI., 1991 b) provides a possible explanation for the paradoxical findings of TIGELAAR and colleagues just summarized. It is possible that the IgEdependent contact hypersensitivity observed by TIGELAAR and colleagues was mediated by y6:TCR+, CD23+ DEC whose FCER were occupied with infused monoclonal IgE anti-DNP antibodies. It is now known that the y6:TCR +, CD23+ cells in the epidermis are «double negative» T cells that do not express CD4 or CDS. The absence of CD4 and CDS on DEC could account for the unexpected persistence of the IgE-dependent hypersensitivity reaction in those mice that had been infused with depleting doses of antiCD4 and anti-CDS monoclonal antibodies. It is conceivable that the IgEdependent contact hypersensitivity reaction observed by TIGELAAR and colleagues is an IgE-dependent ADCC-type reaction. It has been shown by KUZIEL et al. (1991) that DEC have the molecular machinery to mediate ADCC via IgG antibodies and CD16. Our findings and the earlier observations of TIGELAAR raise the possibility of a DEC ADCC reaction mediated via IgE antibodies and CD23. Additional studies are needed to test this hypothesis.

E. Production of soluble Ig-binding factors Another functional significance of FcRs on lymphocytes is that they appear to give rise to soluble, shed forms of the receptors (sFcR) that were originally described by FRIDMAN and colleagues and designated immunoglobulin-binding factors (IBF). The functional significance of sFcyR derived from the IgG FcR on murine lymphocytes has been reviewed (FRIDMAN and SAUTES, 1990). The SFCER derived from the FCER on rodent and human T and B lymphocytes have been investigated in considerable detail and these studies

260 . R. G.

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et al.

have been reviewed (DELESPESSE et aI., 1989; CONRAD, 1990). CONRAD and colleagues have shown that the CD23 released by a B lymphoma appears to be generated by protelytic cleavage of the membrane form of CD23 and that this process can be antagonized by occupancy of the FCER by IgE (LEE et aI., 1987). As discussed above, we have recently found evidence for a CD23 transcript in some T and B lymphocytes that appears to code for a secreted form of CD23 (NUNEZ and LYNCH, 1992). It has been proposed that sFcER is an autocrine B cell growth factor (GORDON et aI., 1985), potentiates IgE production (SAFARTI et aI., 1984; PENE et aI., 1989), and is an MIF-like molecule (FLORES-ROMO et aI., 1989). However, the functional significance of sFcER has remained an area of some debate and additional studies are needed to resolve some of the issues.

VI. Alterations of lymphocyte Fc receptors in disease Since lymphokines, cytokines, immunoglobulins and cellular activation all regulate FcR expression on lymphocytes, it is not surprising that lymphocyte FcR patterns might be altered in certain diseases. As summarized above, mice with plasmacytomas and hybridomas develop large numbers of T lymphocytes that express FcR specific for the heavy chain class of the monoclonal immunoglobulin secreted by the tumor (HOOVER and LYNCH, 1980; MATHUR et aI., 1986; MATHUR and LYNCH, 1986). In mice with tumors that secrete IgG, IgA, IgM or IgE the FcR+ T cells are predominantly CD8+, while with tumors that secrete IgD the FcoR+ cells are predominantly CD4+ (Corco et aI., 1985). This review will focus on the changes in lymphocyte CD23 that occur in disease. In recent studies it was recognized that the B lymphocytes in mice with plasmacytomas exhibited a marked reduction in CD23 (BERG and LYNCH, 1991). The reduction in B lymphocyte CD23 was shown to be mediated by elevated levels of TGF-B produced by plasmacytoma cells and host cells, probably macrophages. This observation was the first demonstration of downregulation of B lymphocyte CD23 by a cytokine. The B lymphocytes from mice with plasmacytomas do not proliferate in response to LPS. However, they do respond with early activation events but become cell cycle arrested late in G1 (BERMAN and ZOLLA-PAZNER, 1987). All of these effects can be generated by incubating normal splenic B cells with TGF-B (MORDUE and LYNCH, unpublished data). These findings raise the possibility that TGF-B plays a role in the severe polyclonal B cell immunodeficiency that occurs in mice with plasmacytomas. It is interesting that CD23, a putative element of the activation apparatus of B lymphocytes, is profoundly downregulated by TGF-B in parallel with the induction of cell cycle arrest. It is not known whether the loss of B cell CD23 contributes to the profound functional impairment of B cells in mice with plasmacytomas, or is simply a marker of TGF-B.


It has been reported that patients with non-allergic hyperimmunoglobulinemia E (Kimura's disease) develop increased numbers of FCER+ T and B lymphocytes (NAGAI et al., 1985). SPIEGELBERG et al. (1985) reported increased numbers of FCER+ lymphocytes in patients with the hyper-IgE syndrome. Similar findings have been made in patients with atopic dermatitis, another disease in which serum IgE levels are markedly increased (NORO et al., 1986). Alterations in T and B lymphocyte CD23 expression have been detected in a number of parasitic infections. Rodents infected with the helminth Nippostrongylus brasiliensis develop high levels of IgE and concomitantly of lymphocytes that express FCER (YODOI and ISHIZAKA, 1979). In murine schistosomiasis the CD4+ T cells in the hepatic granulomas express FcR (SANDOR et al., 1991), a finding that reflects the presence of activated Th2 cells. The CD3+ /yo T cells in the granulomas express FcR, an indication of cellular activation, and the B lymphocytes in the spleen show a 5-fold increase in levels of CD23 expression, which probably reflects elevated IgE and IL-4 (SACCO, SANDOR, WEINSTOCK and LYNCH, unpublished data). In murine malaria, mice infected with Plasmodium chabaudi develop large numbers of T cells that express FcyR and exhibit selective suppression of IgG 1 antibodies (LANGHORNE and TITUS, 1988). In recent studies, NOBEN et al. (1991) found that incubation of the promastigotes of Leishmania donovani with normal murine spleen cells resulted in the selective and complete inhibition of CD23 expression on B lymphocytes. The parasiteinduced loss of B cell CD23 was parasite dose-dependent, requires parasite:lymphocyte contact, and is not reversed by IL-4. Whether and how the loss of CD23 expression on B lymphocytes relates to the polyclonal B cell immune deficiency that occurs in mice with L. donovani awaits further investigation. The selective, parasite-induced decrease in CD23 was also observed on murine and human B cell lines, CLL cells, and on U937, a human monocyte-like cell line. In vivo, CD23 and sIgM were decreased on splenic B cells obtained from mice 24 h after infection with large numbers of leishmania promastigotes. Mice infected with the extracellular protozoan parasite Trypanosoma brucei brucei showed a marked reduction in B cell CD23 and sIgM 3 to 5 days post-infection (R. SACCO, N. NOBEN, M. WILSON, J. DONELSON and R. G. LYNCH, unpublished data). It is likely that alterations in lymphocyte FcR expression in disease will continue to provide useful clues and reliable directions for further investigations. However, the multiplicity of factors that are already known to influence lymphocyte FcR expression make it difficult at this point to sort out cause from effect by simple inspection of the alterations observed. However, a rapidly expanding set of powerful laboratory tools, and a growing body of conceptual insights based on studies in experimental models predict that an understanding of the significance of lymphocyte FcR alterations in disease will be forthcoming.


VII. Coda CD23 is a fascinating element of the immune system. Like its ligand IgE, CD23 is present in rather small amounts and is highly regulated. CD23 appears to participate in a diverse set of important physiological functions. Lymphocyte CD23 is developmentally and environmentally regulated, is differentially expressed on lymphocyte subsets, and is modulated by antigen-specific cellular activation. CD23 is encoded by a single copy gene that contains 12 exons, 11 introns and spans 13 kilobases, and multiple transcripts that encode CD23 isoforms appear to be generated by different transcriptional initiation sites and alternative splicing of RNA. T and B lymphocyte CD23 are altered in many diseases, especially disorders associated with impaired polyclonal B lymphocyte function and/or elevated serum IgE. Most published investigations of CD23 have focused on the structure, regulation and function of B cell CD23. A solid base of descriptive data has been developed, but definitive information about the cell biology and pathology of B cell CD23, while growing, is still rather limited. A particular emphasis of this review has been T cell CD23, an area where, until recently, basic information has been slower to develop, probably because CD23 expression is not constitutive on T cells, the conditions for optimal induction are still being defined, and the quantity of CD23 present is scant. Nonetheless, a multiplicity of findings suggest that CD23 will prove to be an important element of normal and pathologic T cell function. As investigations generate answers to the basic questions about CD23 on T and B lymphocytes, similar issues regarding CD23 on a diverse group of other cells await to be addressed. Acknowledgements We are grateful to ALLAN MUELLER, MARITA ROBINSON, THERESA DUELING, and PATRICK JACOBS for excellent technical contributions, and to VICKI BROWN for excellent secretarial support in the preparation of the manuscript.

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The immunobiology of human Fc gamma receptors on hematopoietic cells and tissue macrophages.

Inhibition of B lymphocyte activation by interaction with Fc receptors.

CD23: possible multiple roles in lymphocyte activation.

CD23: the low affinity receptor for IgE.

Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

MICC cytotoxic effector function of human T lymphocyte subpopulations bearing Fc-receptors for IgG and IgM.

Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.

Role of C'3 and Fc receptors in B-lymphocyte activation.

Expression of the Fc-receptor for IgE (Fc epsilon RII, CD23) on alveolar macrophages in extrinsic allergic alveolitis.

Herpesviral Fc receptors and their relationship to the human Fc receptors.

Comparison of various tests for Fc receptors on different human lymphocyte sub populations.

Independent movement of surface immunoglobulin from Fc receptors on lymphocyte membranes.

Heterogeneity of human lymphocyte Fc receptors. I. Differential susceptibility to proteolysis.

Heterogeneity of human lymphocyte Fc receptors. II. Relationship to antibody-dependent, cell-mediated cytotoxicity.

Expression, regulation and function of human Fc epsilon RII (CD23) antigen.

Soluble CD23 (Fc epsilon RII) and interleukin 1 synergistically induce early human thymocyte maturation.

Human leukocyte IgG Fc receptors.

Functional studies on Fc receptors.

Were lectins primitive Fc receptors?

Fc receptors in human placenta.

The identity of IgE receptors (Fc epsilon RII) that mediate cellular activation of human macrophages: evidence against a role for CD23.

Tumor Fc receptors and tumor-associated immunoglobulins.

CD23+ monocytes in patients with rheumatic diseases.

A range of Cℇ3-Cℇ4 interdomain angles in IgE Fc accommodate binding to its receptor CD23.