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The Journal of Immunology, 1999, 163: 5806-5812.
Copyright © 1999 by The American Association of Immunologists

Crosslinking of the Human Fc Receptor for IgA (Fc{alpha}RI/CD89) Triggers FcR {gamma}-Chain-Dependent Shedding of Soluble CD891

Ger van Zandbergen*, Ralf Westerhuis*, Ngaisah Klar Mohamad*, Jan G. J. van de Winkel{dagger},{ddagger}, Mohamed R. Daha* and Cees van Kooten2,*

* Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; and {dagger} Department of Immunology and {ddagger} Medarex Europe, University Medical Center Utrecht, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD89/Fc{alpha}RI is a 55- to 75-kDa type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. At present, no information is available on the existence of soluble forms of this receptor. We developed an ELISA for the detection of soluble CD89 (sCD89) forms and investigated the regulation of sCD89 production. PMA/ionomycin stimulation of monocytic cell lines (U937, THP-1, and MM6), but not of neutrophils, resulted in release of sCD89. Crosslinking of CD89 either via its ligand IgA or with anti-CD89 mAbs similarly resulted in sCD89 release. Using CD89-transfected cells, we showed ligand-induced shedding to be dependent on coexpression of the FcR {gamma}-chain subunit. Shedding of sCD89 was dependent on signaling via the {gamma}-chain and prevented by addition of inhibitors of protein kinase C (staurosporine) or protein tyrosine kinases (genistein). Western blotting revealed sCD89 to have an apparent molecular mass of 30 kDa and to bind IgA in a dose-dependent fashion. In conclusion, the present data document a ligand-binding soluble form of CD89 that is released upon activation of CD89-expressing cells. Shedding of CD89 may play a role in fine-tuning CD89 immune effector functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunoglobulin A plays a critical role in protecting the host against environmental pathogens and Ags encountered at mucosal surfaces. In humans, IgA is the predominant isotype produced (±66 mg/kg/day) with 80% of all B cells committed to IgA production (1). Receptors for the Fc portion of IgA (Fc{alpha}R) have been identified on a variety of cell types within the immune system and provide a crucial link between the humoral and cellular branches of the immune system (2). Compared with the Fc receptors for IgE (Fc{epsilon}RI and Fc{epsilon}RII) and IgG (Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII), relatively little is known about the nature and function of Fc{alpha} receptors. The best-characterized human Fc{alpha}R described until now, Fc{alpha}RI/CD89, is a type I transmembrane glycoprotein that binds both IgA1 and IgA2 subclasses with similar affinity (Ka ~ 106 M-1) (3). Molecular cloning demonstrated CD89 to be a member of the Ig superfamily (4). The site of interaction between IgA and CD89 was identified on the junction of C{alpha}2 and C{alpha}3 of the IgA molecule (5), and in the membrane distal EC-1 domain of CD89 as shown by both by mutagenesis (3) and domain swapping (6). Comparison of the primary amino acid sequence showed CD89 to be more closely related to killer cell inhibitory receptors than to human Fc{gamma}R (7).

CD89 is constitutively expressed as a 50- to 70-kDa protein on neutrophils and monocytes/macrophages, or as a 70- to 100-kDa glycoprotein on eosinophils due to increased glycosylation (2, 8). The CD89 molecule is associated through a charge-based mechanism with the common FcR {gamma}-chain, which connects CD89 to intracellular signaling pathways via immunoreceptor tyrosine-based activation motifs located within the cytoplasmic tail of the FcR {gamma}-chain (9, 10). Crosslinking of CD89 on myeloid cells can trigger diverse processes including phagocytosis, superoxide generation, Ab-dependent cellular cytotoxicity, and release of inflammatory mediators (2).

Several signals have been shown to modulate surface expression of CD89. Cytokines (TNF-{alpha}, GM-CSF, IL-1ß, IL-8), LPS, PMA, and aggregated IgA can induce increased CD89 expression on cells (8, 11, 12). In contrast, TGF-ß (13) and suramin (14) were shown to down-regulate its expression. Altered CD89 expression may directly affect the effector function of CD89-expressing cells. Soluble forms have been identified for various Fc receptors for IgG (Fc{gamma}RII/CD32 and Fc{gamma}RIII/CD16) and IgE (Fc{epsilon}RII/CD23) (15, 16, 17). Furthermore, it has been proposed that these soluble Fc receptors have a pathophysiological role in several diseases (18, 19, 20). No information is available on the existence of soluble forms of Fc{alpha}R.

In the present study we demonstrate the existence of a soluble CD89 (sCD89)3 protein, a 30-kDa glycosylated protein with retained ability to bind human IgA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of recombinant CD89 protein and anti-CD89 reagents

A recombinant soluble form of CD89 was produced by expressing the cDNA encoding the extracellular part of CD89 (21) in CHO-K1 cells using the pEE14 expression system (Celltech, Slough, U.K.). The stable CHO-K1 transfectant produced ~15 µg/ml of recombinant sCD89. Using columns of immobilized IgA, more than 99% of the recombinant soluble protein was recovered from culture supernatants (22). The purity of the preparations was checked by SDS-PAGE, and a single band was detected by Coomassie brilliant blue staining. The recombinant sCD89 protein was used to immunize mice, a rabbit, and a goat. CD89-reactive rabbit and goat antisera were raised and used as purified IgG fractions. Using standard hybridoma technology we raised novel mouse mAbs specific for CD89. The specificity of these reagents was confirmed by FACS analysis on CD89-transfected cells and by immunoprecipitation and Western blotting (6, 23).

ELISA for sCD89

Rabbit anti-CD89 IgG (2 µg/ml) was coated to ELISA plates (NUNC Maxisorb, Life Technologies, Gaithersburg, MD) by overnight incubation at room temperature in coating buffer (0.1 M NaHCO3/Na2CO3, pH 9.6). The wells were washed three times using washing buffer (PBS, 0.02% Tween 20) and, subsequently, varying concentrations of the recombinant sCD89 protein or BSA (as a control) were added. All samples were diluted in ELISA buffer (PBS, 0.02% Tween 20, 1% FCS) and incubated for 1 h at 37°C. Following incubation, wells were washed as above and incubated first with digoxigenin (Dig)-conjugated Rabbit F(ab')2 anti-CD89 (1 µg/ml), followed by HRP-conjugated F(ab')2 anti-Dig (1/5000, Boehringer Mannheim, Indianapolis, IN) (both for 1 h at 37°C and washed in between as above). OD415 was measured after addition of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)/H2O2 as substrate. The optical density at 415 nm was assessed using a Microplate Biokinetics Reader EL 312e (Bio-Tek, Burlington, VT).

We also used a sandwich ELISA of monoclonal and polyclonal Abs for the quantification of CD89 in supernatants of PMA/ionomycin-activated cells and found similar values as measured in an ELISA with polyclonal Ab coating. However, because four of five mAbs recognize the IgA binding site on CD89 (6), this hampers the study of IgA- or anti-CD89-induced shedding. Therefore, for consistency in our work, we have chosen to present all data from one type of ELISA with which we used the polyclonal Ab as a coating.

Soluble CD16 was measured by ELISA (24).

Cell culture and activation

Polymorphonuclear leukocytes (PMNs) and monocytes were isolated from whole blood of healthy donors by Ficoll density centrifugation. The following CD89-expressing cell lines were used: U937 (ATCC nr CRL-1593.2) (25), THP-1 (ATCC nr TIB-202) (26), and MonoMac-6 (kindly provided by Dr. H. W. L. Ziegler-Heitbrock, Institut fur Immunologie, Universitat Munchen, Munchen, Germany) (27). All cells were cultured at 37°C with 5% CO2 in a humidified atmosphere in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies). IIA1.6 cells were grown in the same medium supplemented with geneticin (G418, 0.8 mg/ml; Life Technologies) for CD89-transfected cells, or geneticin and methotrexate (10 mM; Pharmachemie, Haarlem, The Netherlands) for cells cotransfected with CD89 and FcR {gamma}-chain (10). Cell viability was greater than 95% for all cell preparations used.

For activation, PMNs were cultured at a concentration of 1.0 x 107/ml (24). Monocytes and myeloid cell lines were activated at a concentration of 2.0 x 106 cells/ml. All activation experiments were performed in triplicate. After the indicated times, cells were harvested and tested by FACS analysis or supernatants were harvested and tested by ELISA. The following stimuli were used: PMA (10 ng/ml), ionomycin (1 µg/ml), and LPS (Salmonella thyphosa, 100 ng/ml) (all from Sigma, St. Louis, MO). In addition, various purified IgA preparations isolated from normal human serum, sera from myeloma patients, anti-CD89 mAbs, and goat anti-mouse Ig Abs were used (all prepared in our laboratory) (6, 22, 23, 28).

For inhibition of {gamma}-chain-induced signal transduction, we have used inhibitors of protein kinase C (staurosporine; 50 ng/ml) or protein tyrosine kinases (genistein; 100 µM) (both from Sigma). These concentrations were nontoxic for the cells as determined by trypan blue exclusion.

FACS analysis

For FACS analysis, cells (5 x 105) were incubated with the CD89 mAb 2D11 (IgG1) or an isotype-matched control, diluted in FACS buffer (PBS/0.5% BSA/0.02% azide). After incubation for 1 h at 4°C, cells were washed with FACS buffer and incubated for 1 h with PE-conjugated goat anti-mouse IgG1 polyclonal IgG (Southern Biotechnology Associates, Birmingham, AL). After washing, cells were fixed with 1% paraformaldehyde in PBS and analyzed on a FACScan (Becton Dickinson, San Jose, CA). Data acquisition and analysis were conducted with Lysis II (Becton Dickinson).

Immunoprecipitation and Western blotting

As a positive control for intact CD89, U937 cells (1 x 108) were lysed using PBS/1% NP40. Cell lysates and culture supernatants were separated on 10% SDS polyacrylamide gels under reducing conditions and blotted onto polyvinylidene difluoride membrane (Millipore, Bedford, MA). Using standard Western blotting protocols, different forms of CD89 were detected with a mixture of rabbit and goat IgG anti-CD89 (both 10 µg/ml). After incubation and washing, followed by subsequent incubation with HRP-conjugated swine anti-rabbit IgG (1/50,000; Dako, Denmark) and HRP-conjugated rabbit anti-goat IgG (1/50,000; Dako). Signals were visualized using Super Signal Chemiluminescence substrate, according to manufacturers’ instructions (Pierce, Rockford, IL).

Isolation of sCD89- and IgA-binding ELISA

The sCD89 protein was isolated from culture supernatant of PMA/ionomycin-stimulated U937 cell using an affinity column of human IgA isolated from normal serum (22). Preparations of purified IgA from normal human serum (2 µg/ml) were coated to an ELISA plate and binding of sCD89 was detected using Dig-conjugated Rabbit F(ab')2 anti-CD89, similar to the CD89 ELISA described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of an ELISA for sCD89

To study the presence of sCD89, we developed a CD89-specific ELISA, using Rabbit polyclonal Abs. As a positive control, a recombinant sCD89 protein produced in CHO cells was employed. Increasing concentrations of recombinant sCD89 resulted in a dose-dependent signal in this ELISA. The detection limit of this ELISA was reproducibly found to be ~50 pg/ml (Fig. 1Go).



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  		FIGURE 1. Detection of sCD89 protein by ELISA. Rabbit anti-CD89 IgG (2 µg/ml) was coated to ELISA plates, and samples containing different concentrations of either recombinant sCD89 ({triangleup}) or BSA ({blacksquare}) were added. After incubation, wells were washed, and bound CD89 was detected with Dig-conjugated rabbit F(ab')2 anti-CD89 (1 µg/ml), then with HRP-conjugated F(ab')2 anti-Dig. OD415 was measured after addition of ABTS/H2O2 as substrate. The detection limit of sCD89 ELISA was reproducibly found to be 50 pg/ml (extinction background +2x SD in six independent experiments).

 
Monocytic cells release sCD89 upon activation

We investigated the release of sCD89 in supernatants of activated human cells. First, the release of CD89 was studied in the pro-monocytic cell line U937, a cell type expressing high levels of CD89. Stimulation of U937 with PMA and ionomycin consistently induced release of a sCD89 (Fig. 2GoA). sCD89 was first detectable after 3 h and reached a maximum at 36 h. In five independent experiments the amount of sCD89 detected ranged from 25 to 43 ng/ml. When the same number of U937 cells were cultured in the absence of PMA/ionomycin, no sCD89 could be detected (Fig. 2GoA).



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  		FIGURE 2. Detection of sCD89 in supernatants of PMA/ionomycin-stimulated cells. A, U937 cells were cultured in medium alone ({triangleup}) or in medium supplemented with PMA/ionomycin ({blacktriangleup}). At indicated time points supernatants were tested for sCD89. Results are expressed as the mean ± SEM of three independent experiments. B, Freshly isolated PMNs were cultured in medium with PMA/ionomycin. At the indicated time points supernatants were harvested and tested for sCD89 ({blacktriangleup}) and sCD16 ({circ}). Results are expressed as the mean ± SEM of three independent experiments, using cells from three different donors.

 
Next PMNs, cells that expresses amounts of CD89 similar to U937, were stimulated with PMA and ionomycin. Irrespective of the time points tested, which varied from 1 min to 36 h, sCD89 could not be detected in the supernatants (Fig. 2GoB). As a positive control for the activation conditions, the same supernatants were tested for the release of CD16. In accordance with the literature (16, 24), after 1 min soluble CD16 was detectable in supernatants of activated PMN, and this production further increased over time (Fig. 2GoB).

Regulation of CD89 surface expression

Because modulation of surface expression might contribute to the release of CD89, we investigated the effect of PMA/ionomycin on CD89 membrane expression as detected with mAbs. FACS analysis showed that PMA/ionomycin induced a 3- to 10-fold increase (range of four independent experiments) in CD89 surface expression on U937 cells compared with cells cultured in medium alone (Fig. 3Go). This up-regulation was not unique for PMA/ionomycin and could also be observed with other stimuli that have previously been found to affect CD89 expression (12). Both LPS and heat-aggregated human IgA (aIgA) enhanced CD89 surface expression on U937 cells.



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  		FIGURE 3. FACS analysis of CD89 surface expression. U937 cells were cultured in medium alone or in medium supplemented with PMA/ionomycin, LPS, or aIgA. After a 36-h culture, CD89 surface expression was assessed with CD89 mAb 2D11 (1/1000) then with PE-conjugated Goat anti-mouse-IgG1. Hatched histograms represent conjugate controls; open histograms represent CD89 expression. Of three independent experiments, one histogram is shown per condition. The dotted line represents the median fluorescence for CD89 expression of cells cultured in medium alone.

 
Crosslinking of CD89 by IgA or anti-CD89 Abs induces release of a soluble form

To verify whether the release of sCD89 is correlated with up-regulated surface expression, supernatants of stimulated U937 were tested. In addition to PMA/ionomycin, high m.w. forms of both IgA1 and IgA2 (polymeric or heat aggregated) consistently induced release of sCD89 from U937 cells (Fig. 4GoA). In contrast, little activation was observed with preparations containing monomeric IgA. No sCD89 was detected after LPS stimulation of U937 (Fig. 4GoA).



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  		FIGURE 4. CD89-specific crosslinking triggers sCD89 release from U937. A, U937 cells were cultured in medium alone or in medium supplemented with PMA/ionomycin, LPS, or aIgA2 from a patient with an IgA2 myeloma, polymeric IgA1 (pIgA1) from a patient with an IgA1 myeloma, or monomeric serum IgA (mIgA), as indicated. After 36 h of culture, supernatants were collected and tested for sCD89. Results are expressed as the mean ± SEM of three independent experiments. B, U937 cells were cultured in medium supplemented with different concentrations of the anti-CD89 mAb 2D11 ({blacktriangleup}) or an isotype-matched control ({triangleup}). After 36 h, supernatants were collected and tested for sCD89. Results are expressed as the mean ± SEM of three independent experiments.

 
We then tested Fc{alpha}RI-crosslinking using different concentrations of the anti-CD89 mAb 2D11 and found a dose-dependent induction of sCD89 release (Fig. 4GoB). An isotype-matched control Ab did not induce the release of CD89, whereas crosslinking 2D11 using Goat anti-mouse IgG Abs increased CD89 shedding.

Similar activation conditions were applied to freshly isolated peripheral blood monocytes and two other myeloid cell lines, MonoMac-6 and THP-1. Activation with PMA/ionomycin or with IgA stimulated an increased surface expression on all three cell types (data not shown). In addition, both PMA/ionomycin and aIgA, as well as anti-CD89 Abs, induced the release of sCD89 (Fig. 5Go).



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  		FIGURE 5. CD89 release from monocytic cell lines. Peripheral blood monocytes (A), MonoMac-6 (B), and THP-1 (C), all of which are CD89-expressing cells, were cultured in medium alone or in medium supplemented with PMA/ionomycin, LPS, aIgA2, or anti-CD89, as indicated. After 36 h, supernatants were collected and tested for sCD89. Detection limit is shown (100 pg/ml; thin dotted line in A). Results are expressed as the mean ± SEM of three independent experiments.

 
The common FcR {gamma}-chain is essential for CD89-triggered release of sCD89

To study the mechanism of CD89-triggered release of sCD89 in more detail, we used murine IIA1.6 cells transfected with human CD89 alone, or with human CD89 in combination with the FcR {gamma}-chain subunit. Both transfectants were previously shown to have a comparable CD89 expression, and both cell lines displayed a similar IgA binding (29). Activation of CD89/{gamma}-chain transfected cells with increasing amounts of aIgA triggered a dose-dependent release of sCD89. No sCD89 could be detected in supernatants of aIgA-stimulated cells transfected with CD89 alone (Fig. 6GoA). Similarly, anti-CD89 Abs induced release of sCD89 only in cells co-expressing the {gamma}-chain (Fig. 6GoB). PMA/ionomycin stimulation led to release of sCD89 in both cell types (Fig. 6GoB). To investigate whether signaling via the {gamma}-chain is important for the release of sCD89, two specific inhibitors were used. Addition of either an inhibitor of protein kinase C (staurosporine) or an inhibitor of protein tyrosine kinases (genistein) prevented the shedding of sCD89 from the surface of aIgA-stimulated IIA1.6 CD89/{gamma} chain transfectants or U937 cells (Fig. 7Go).



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  		FIGURE 6. Release of CD89 from transfected cells is dependent on FcR {gamma}-chain. A, CD89-transfected cells were cultured in medium supplemented with varying concentrations of aIgA2. After 36 h, supernatants of stimulated IIA1.6 CD89/{gamma} ({blacksquare}) and IIA1.6 CD89 ({circ}) cells were collected and tested for sCD89 protein. Results are expressed as the mean ± SEM of three independent experiments. B, CD89-transfected cells were cultured in medium alone, or in medium with PMA/ionomycin or with anti-CD89 mAb 2D11 and 7D7. After 36 h, supernatants of the stimulated IIA1.6 CD89/{gamma} ({blacksquare}) and IIA1.6 CD89 ({square}) cells were collected and tested for sCD89 protein. Results are expressed as the mean ± SEM of three independent experiments.

 


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  		FIGURE 7. Shedding of sCD89 is prevented by genistein and staurosporine. Both the CD89/{gamma} transfectants (left) and the U937 cells (right) were cultured stimulated with aIgA2 with or without an inhibitor of protein tyrosine kinases (gen., genistein; 100 µM) or an inhibitor of protein kinase C (stau., staurosporine; 50 ng/ml). After 36 h, supernatants were collected and tested for sCD89 protein. Of three independent experiments, one representative example is shown.

 
Biochemical characterization of sCD89

To further analyze the nature of sCD89, Western blotting was performed on cell lysates and supernatants of myeloid cells. Utilizing rabbit and goat polyclonal anti-CD89 Abs, a broad band ranging from 55 to 75 kDa was detected on U937 cell lysates (Fig. 8GoA, lane 2). When analyzing the supernatants of U937 cells, a specific product of ~30 kDa was found after PMA/ionomycin stimulation, which was not observed under nonstimulated conditions (lanes 3 and 4). Reactivity against the 30-kDa protein was completely blocked by preincubating the antisera with recombinant sCD89 (lane 5). A similar 30-kDa protein was found in the supernatant of PMA/ionomycin-stimulated THP-1 cells (lanes 6 and 7) and in supernatant of aIgA-stimulated CD89/{gamma}-transfected IIA1.6 cells (lanes 8 and 9).



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  		FIGURE 8. Immunochemical analysis of sCD89 protein. A, Cell lysates from U937 cells were blotted with normal rabbit and goat sera as a control (lane 1), or with CD89-specific antisera (lane 2). Supernatants of U937 and THP-1 cells cultured in medium alone or in culture stimulated for 36 h with PMA/ionomycin (as indicated) were blotted with CD89-specific sera (lanes 3, 4, 6, and 7, respectively). As a specificity control (lane 5), rabbit and goat polyclonal anti-CD89 Abs were preincubated with 25 µg of recombinant sCD89 and used for blotting of supernatant from PMA/ionomycin-stimulated U937 (P/I*). Similarly, U937 and THP-1 supernatants of IIA1.6 CD89/{gamma} were cultured in medium alone or in culture stimulated with aIgA2 and blotted (lanes 8 and 9, respectively). B, Purified sCD89 was tested at the indicated concentrations for binding to IgA (coated 2 µg/ml, {blacktriangleup}) or BSA (coated 2 µg/ml, {square}) as a specificity control. Results are expressed as the mean ± SEM of three independent experiments. C, Recombinant sCD89 and purified sCD89 were treated with N-glycosidase F for removal of N-linked sugars. Deglycosylated or untreated samples, as indicated, were separated on 10% SDS-PAGE and blotted specificly for CD89.

 
To obtain more detailed information on the structure of sCD89, the protein was purified from supernatant of PMA/ionomycin-activated U937 cells. sCD89 could be purified using an IgA-affinity absorbent, which showed the natural form of sCD89 to be capable of binding IgA. This was confirmed in an ELISA system, with IgA coated to ELISA wells, where a dose-dependent binding of sCD89 to IgA was observed (Fig. 8GoB). When the purified sCD89 was analyzed by Western blotting, we observed a 30-kDa molecule. Treatment with N-glycanase reduced the molecular mass to 25 kDa. For comparison, the recombinant soluble form (37–55 kDa) was treated in the same way and predictably showed a 27-kDa backbone after removal of the N-linked glycans (Fig. 8GoC) (21).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrate that upon activation, myeloid cells can release a soluble form of Fc{alpha}RI/CD89. Biochemical analysis showed this soluble receptor to represent a 30-kDa glycosylated protein that is capable of binding IgA. Both aIgA and anti-CD89 Abs induced the release of sCD89, which suggests that IgA Abs produced during a mucosal immune response might have a regulatory effect on the CD89 effector functions.

In our experiments we concentrated on the myeloid cell line U937, although similar effects were found with other monocytic cell lines (THP-1 and MonoMac6). Activation of PMN with PMA/ionomycin results in a strong and fast release of sCD16, but does not result in release of sCD89, suggesting that regulation of receptor-shedding is different between CD16/Fc{gamma}RIII and CD89/Fc{alpha}RI. Comparing peripheral blood monocytes and myeloid cell lines, we found that the regulation of sCD89 shedding was qualitatively similar. However, in peripheral blood monocytes we detected lower amounts of sCD89 in our ELISA. This might be partially explained by a low-level expression of CD89 on monocytes or by differences in regulation between these myeloid cell lines and monocytes. We showed that the release of sCD89 is dependent on an active signaling event, which might be quantitatively different in cell lines. It was demonstrated that release of CD89 after CD89 crosslinking is dependent on the presence of the common {gamma}-chain, which is also associated with Fc{epsilon}RI, Fc{gamma}RIIIa/CD16, and Fc{gamma}RIIa/CD32 (2) (Fig. 6GoA). Signaling via this subunit induces protein kinase C activation (9), as well as tyrosine phosphorylation of the {gamma}-chain by members of the Src family (Lyn, Syk), phosphatidylinositol-3 kinase activation, and Bruton tyrosine kinase activation (30, 31). Accordingly, we were able to block the release of sCD89 by inhibition of protein kinase C or protein tyrosine kinases.

In contrast to sCD16 shedding (16), release of sCD89 was rather slow, suggesting the involvement of secondary processes. Induction of sCD89 release was accompanied by up-regulation of surface expression, although increased surface expression did not always result in sCD89 shedding. Previous experiments have shown that the IIA1.6 transfectants we have used in our experiments have a comparable CD89 expression and display similar IgA binding (32); therefore, the presence of the {gamma}-chain seems to have no effect on the affinity for IgA. These findings are different from data published for Fc{gamma}R (33) and require further investigation.

The molecular mass of 30 kDa of CD89 rules out the possibility that the products measured in ELISA are released membrane vesicles containing full-length CD89. Recently, at least 11 different splice variants of CD89 have been identified (34, 35, 36, 37, 38). It seems unlikely that they are responsible for the sCD89 molecule because most of them showed partial or complete deletions of EC1 or EC2, but still contained the predicted transmembrane region. Finally, we found that IIA1.6 cells transfected with full length CD89 cDNA, excluding alternative splicing, also release a similar 30-kDa molecule upon activation (Fig. 7GoA, lane 8). These data suggest a role for proteolytic cleavage, as demonstrated for various molecules including cytokines (TNF-{alpha}), cytokine receptors, adhesion molecules, and Fc receptors (18, 24, 39). After C-terminal sequencing large amounts of sCD16 purified from human serum, the cleavage site of CD16/Fc{gamma}RIII was identified as being between Val196 and Ser197 (40). In preliminary experiments we found that both EDTA and 1,10 phenantriolin, which are inhibitors of metalloproteinases, prevented the release of sCD89 (data not shown), suggesting the involvement of metalloproteinases in cleavage of CD89. The difference in core size between recombinant sCD89 and sCD89 cleaved from U937 shows that the cleavage site is N-terminal from Tyr207, the C-terminal amino acid of the recombinant product.

An important question concerns the (patho-) physiological role of sCD89. Release of soluble receptors has been suggested to represent a universal mechanism of receptor regulation, which might be dysregulated in various human diseases (18). Shedding of CD89 will uncouple the receptor from its signaling transduction pathways and, therefore, it represents a means of effector function down-regulation. Quantification of CD89 in cell lysates of U937 compared with their supernatants suggested that up to 5% of the receptor might appear in soluble form after stimulation with PMA/ionomycin (data not shown). It is likely that sCD89 immediately interact with circulating IgA and influence the function of IgA. We have obtained preliminary evidence that CD89 is present in the circulation. It is possible that IgA-CD89 complexes have "nephritogenic" activities as has been suggested recently (41).

Levels of sCD16 (Fc{gamma}RIII) have been proposed to be a measure for the number of neutrophils (16). Our in vitro data suggest that PMNs do not release CD89 and that monocytes might be the most important source of sCD89. Therefore, sCD89 levels might represent a measure for monocyte numbers and/or activation. Recently, monocytes (but not neutrophils) of patients with primary IgA nephropathy, were found to display a marked reduction of surface CD89 expression that correlated with the increased levels of serum IgA (42). At present it is unclear whether the negative regulation of monocytic CD89 expression is associated with an increased release of sCD89.

In conclusion, we have shown that the myeloid Fc{alpha}RI/CD89 can be released as a 30-kDa soluble molecule. The release of sCD89, which can bind IgA, is induced upon activation of myeloid cells. This may provide a mode of "fine-tuning" effector functions of CD89 expressing cells. In recent years the CD89 molecule has evolved as a candidate target for bispecific Ab therapy (43, 44). It will be important to unravel the mechanisms of CD89 shedding, not only to potentially improve the efficacy of therapy, but also to monitor immune activation.


   Acknowledgments
 
We thank Dr. P. J. Middelhoven (Central Laboratory of the Netherlands Red Cross Blood Transfusion, Amsterdam) for performing the soluble CD16 ELISA and Dr. P. Hiemstra (Department of Pulmonology, Leiden University, Leiden, The Netherlands) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the Netherlands Organization for Scientific Research Grant 901-12-214. C.v.K. is a fellow of the Royal Dutch Academy of Sciences. Back

2 Address correspondence and reprint requests to Dr. C. van Kooten, Department of Nephrology, Leiden University Medical Center, Building 1, C3P, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. E-mail address: Back

3 Abbreviations used in this paper: sCD89, soluble CD89; Dig, digoxigenin; PMN, polymorphonuclear cell; aIgA, heat-aggregated human IgA; ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Back

Received for publication April 26, 1999. Accepted for publication September 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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