Introduction

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Like mast cells, basophils secrete mediators in response to antigenic stimulation of cell-surface immunoglobulin (Ig) E linked to its high-affinity receptor. Basophils are found in airway tissues and fluids during allergic reactions associated with asthma and are therefore thought to participate in the maintenance of these reactions. When considering the general population, IgE-mediated mediator release from peripheral blood basophils varies considerably (1). Some subjects have basophils that completely fail to release mediators in response to stimulation with anti-IgE antibodies, antireceptor antibodies, or antigens (on cells passively sensitized with antigen-specific IgE) (2). These basophils have cell-surface densities of IgE and receptor that are equivalent to basophils that secrete, and IgE-mediated stimuli can be demonstrated to bind to the cell-surface IgE (2, 3) and induce changes in basophil function without secretion. In addition, basophils with the nonreleasing phenotype can respond to stimuli other than anti-IgE, such as the bacterial peptide formylmethionyl leucylphenylalanine (fMLP), indicating that receptor-induced degranulation or mediator generation can occur (2, 3).

The high-affinity IgE receptor belongs to a family of multichain immune-system receptors that includes the T-cell receptor, the mIg receptor of B-cells, and the high (FcRI)- and low (FcRIII/CD16)-affinity Fc receptors for IgG. These receptors all lack intrinsic tyrosine kinase activity (4). Instead, they recruit and activate cytoplasmic tyrosine kinases, which in turn phosphorylate tyrosine residues in characteristic receptor subunit sequences called immunoreceptor tyrosine-based activation motifs (ITAMs) (5). As found in animal mast-cell models, we and others have demonstrated in the human basophil that protein tyrosine kinase activation is an early event after FcRI crosslinking (8, 9). Among the recruited tyrosine kinases are lyn and syk which, in the RBL-2H3 cell line, have been shown to be preferentially associated with the ITAMs present within the  and  subunits of the IgE receptor, respectively (10). Studies by Yamaguchi and colleagues demonstrated that the high-affinity IgE-receptor on nonreleasing human basophils does not contain a mutation in either the , , or  subunits (13). However, recent studies by Kepley and Andrews have suggested that nonreleasing human basophils lack syk kinase (14). In the present study, we present data that confirm the recent studies by Kepley and Andrews concerning syk expression levels (14), extend the observation to lyn kinase expression levels, and present data that suggest that this condition exists while the cells express normal levels of messenger RNA (mRNA) for both lyn and syk kinases.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

fMLP, phorbol 12-myristate 13-acetate, human IgG, piperazine- N,N'-bis-2-ethane sulfonic acid (Pipes), glucose, ethyleneglycol- bis-N,N,N',N'-tetraacetic acid, fetal calf serum, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), human serum albumin (HSA), sodium orthovanadate, benzamidine, aprotinin, phenylmethylsulphonyl fluoride (PMSF), sodium fluoride, 2-mercaptoethanol, nonidet P-40 (NP-40), and accuspin tubes were all purchased from Sigma Chemical Co. (St. Louis, MO). Sodium dodecyl sulfate (SDS) Tween and Tris were purchased from Bio-Rad (Hercules, CA). Agarose was purchased from GIBCO BRL (Gaithersburg, MD). Protein G sepharose and Percoll were purchased from Pharmacia Biotec (Piscataway, NJ). The 4-20% Tris-glycine gels and 2× sample buffer were bought from Novex (CA); and biotinylated molecular-weight markers were purchased from New England Biolabs (Beverly, MA). The antibody cocktail and columns used in the negative selection of human basophils were purchased from Miltenyi Biotech (Auburn, CA), as were the glycophorin A microbeads. Mouse antihuman p72syk (4D10) (recognizes amino acids 313-339 within the linker region), rabbit antihuman p72syk (epitope mapping within the linker region), and rabbit antihuman p53/56lyn (recognizes amino acids 44-63 within the amino-terminal domain) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse antihuman Btk (recognizes amino acids 25-173 of human Btk) was purchased from Pharmingen (Torrey Pines, CA). Sheep antimouse Ig horseradish peroxidase (HRP), streptavidin HRP conjugate, enhanced chemiluminescence (ECL) Western blotting detection Agents, and ECL hyperfilm were all purchased from Amersham (Buckinghamshire, UK). Goat antihuman IgE was prepared as described previously; the antibody used for these studies represented the IgG fraction of goat serum prepared by DE-52 chromatography (15). All other reagents used were of the highest grade available.

Buffers. Pipes buffer contained 25 mM Pipes, 110 mM NaCl, and 5 mM KCl adjusted to pH 7.4 with 1 N HCl; Pipes-albumin- glucose (PAG) also contained 0.003% (wt/vol) HSA and 0.1% (wt/vol) glucose; PAGCM was supplemented with 1 mM CaCl₂ and 1 mM MgCl₂. Column buffer used in the negative selection of human basophils contained Pipes, 0.5% BSA, and 2 mM EDTA. Lysis buffer contained 20 mM Tris (pH 7.8), 150 mM sodium chloride, 1% NP-40, 5% glycerol, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM benzamidine, and 1 µg/ml aprotinin. In the electrophoresis studies, 2× sample buffer contained 0.5 M Tris-HCl (pH 6.8), 10% (wt/vol) SDS, 0.1% bromophenol blue, 20% glycerol, and 5% mercaptoethanol; TBST buffer contained 12 mM Tris base (pH 7.5), 150 mM NaCl, and 0.05% Tween-20; running buffer contained 25 mM Tris base, 192 mM glycine, and 0.1% SDS; transfer buffer contained 12 mM Tris base, 96 mM glycine, and 20% methanol; stripping buffer contained 7 M guanidine hydrochloride.

Subjects

Donors consisted of adult subjects who were randomly selected from the personnel of the Department of Clinical Immunology. The donors were mostly nonatopic as assessed by questionnaire. For the purpose of this study, histamine release from human basophils in response to anti-IgE (0.2 µg/ml) antibody was determined on at least three different occasions. A "nonreleaser" was defined as a donor whose basophils released < 5% histamine, whereas a "releaser" was a donor whose basophils released > 10% histamine on all three occasions. Donors were asked if they had any medical problems, especially any known history of atopy (asthma, allergic rhinitis, chronic urticaria, atopic dermatitis, etc.), and if they were receiving any medication.

Isolation of Human Basophils

Blood was drawn by venipuncture, and the cells were separated using a two-step Percoll density gradient and negative selection as previously described (16, 17). Briefly, the leukocytes were partially purified in accuspin tubes on a two-step Percoll density gradient (1.070/ 1.082 g/ml). The interface layer between the two Percoll layers was removed and washed in PAG and with all donors was found to contain 99 ± 3% of the total number of basophils at a purity of 10 to 20%. The basophils were then resuspended in column buffer and further purified by negative selection using the Miltenyi basophil isolation kit (which contains a cocktail of hapten-conjugated CD3, CD7, CD14, CD15, CD16, CD36, CD45RA, and human leukocyte-associated antigen-DR antibodies and magnet-activated cell separation microbeads coupled to an antihapten conjugated monoclonal antibody) and glycophorin A microbeads. The ranges of basophil yield for both groups of donors were identical and any remaining contaminating cells consisted mainly of monocytes and lymphocytes.

Cell Counting

Basophils were stained with Alcian blue and counted in a Spiers Levy hemocytometer (18). For all these studies, basophil purities were 97 to 100%. Viability was also assessed by trypan blue exclusion and for these studies basophil viability was > 95%.

Cell Stimulation and Histamine Release

Human basophils (2 × 10⁴) were resuspended in PAGCM and then challenged with either goat antihuman IgE antibody (0.2 µg/ml) or formyl methionyl leucyl phenylalanine (fmet) peptide (1 µM) for 45 min at 37°C. After 45 min, all reactions, performed in duplicate, were stopped by centrifugation and the supernatant was removed for histamine analysis. In each experiment, perchloric acid at a 1.6% final concentration was added to some tubes to determine total histamine content. Histamine was assayed by an automated fluorometric technique (19). The percentage of histamine release was calculated from the ratio of sample to total histamine after spontaneous release was subtracted from both.

Lysate Preparation

Purified human basophils (> 97%) were counted as described earlier, then directly spun down (14,000 × g for 10 s), and lysed in 1× sample buffer at a final concentration of 25 × 10⁶ per ml, then boiled at 100°C for 5 min. Samples were then stored at 130°C until analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting. A positive control for p53/56lyn and p72syk was also prepared from a mononuclear cell preparation, aliquotted (to minimize freeze-thaw effects), and stored at 130°C.

Immunoprecipitation

For some preparations, immunoprecipitation was also performed. Briefly, centrifuged lysates were precleared with protein G sepharose beads for 30 min at 4°C. The precleared lysates were then washed and incubated with 1 µg/ml anti-p72syk prebound to protein G sepharose beads. After gentle rotation for 1 h at 4°C, the beads were washed and the immunoadsorbed proteins were eluted from the beads by boiling in 2× SDS sample buffer. Control experiments revealed that an irrelevant IgG antibody or mouse antihuman p72syk in the absence of lysate did not pull down syk in the immunoprecipitates.

SDS-PAGE and Western Blotting

Proteins were separated in a 4-20% Tris-glycine gel under reducing conditions and electrotransferred onto a nitrocellulose membrane. The free binding sites were blocked by incubating the membrane overnight at 4°C with 4% BSA in TBST. The nitrocellulose membranes were then incubated with either 0.2 µg/ml antihuman p72syk, 0.2 µg/ml antihuman p53/56lyn, or 1 µg/ml antihuman Btk for 1 h at room temperature. The membrane was then washed with TBST before the addition of an antimouse or antirabbit HRP conjugate (1:3,000 dilution) for 1 h at room temperature. After further washing of the membranes with TBST the proteins were visualized using ECL. The nitrocellulose membrane was exposed to ECL hyperfilm for various times ranging from 15 s to 5 min, a time course previously determined to provide exposures that yielded reasonably linear quantification of syk and lyn protein. In all experiments, equal protein loading was determined by Coomassie staining.

Quantification of Protein Levels

After exposure to chemiluminescence detection agents, the intensity of each band was determined by densitometric analysis with a Kodak DC120 digital camera and acquisition software. The relevant expression levels of p72syk and p53/56lyn in the basophil were then quantified as a fraction of a positive control run at the same time. In pilot experiments, we determined from 2-fold dilutional analysis that ECL detection was linear over the range of detection required. To verify repeatability, some samples were run in duplicate in the same blot and found to give ratios to the standard that were within the range of ± 0.01. In addition, some samples were run in different blots (but with the same standard) and found to yield similar standard ratios that were within the range of ± 0.03. It should also be noted that our antibodies against p72syk and p53/ 56lyn detect both alternatively spliced forms of syk and lyn.

mRNA Expression

Total RNA was isolated from highly purified human basophils (> 99%) by RNAzol (Tel Test, Inc., Friendswood, TX) according to the manufacturer's instructions. Briefly, 0.5 × 10⁶ basophils were resuspended in 0.6 ml RNAzol. The total mRNA was then extracted by a chloroform/isopropanol technique. Any contamination was then removed by an ethanol wash. Dried ethanol precipitates were resuspended in diethylpyrocarbonate-treated water. A positive control for p53/56lyn and p72syk was also prepared from a mononuclear cell preparation and aliquotted to minimize freeze-thaw effects. All samples were stored at 130°C until mRNA was quantified using Real Time polymerase chain reaction (PCR).

Real Time PCR

Forward and reverse oligo primers for p72syk and p53/56lyn were constructed using Primer Express software and previously published complementary DNA (cDNA) sequences (20, 21). For p72syk the forward primer was 5'-AGCAGAAGCAAATGTCATGCA-3' (nucleotide position 1,401, Genbank accession no. L28824) and the reverse primer 5'-CCTCGCATATCCCGATCATC-3'. For p53/56lyn the forward and reverse primers were 5'-GCAGAGGGAATGGCATACATC-3' (nucleotide position 1,014, Genbank accession no. M79321) and 5'-TGCAAGGCCAAAATCTGCAA-3', respectively. The primers were designed to detect both forms of syk and lyn. The probes for use in the Real Time PCR machine were also designed using Primer Express and the previously reported cDNA sequences for p53/ 56lyn and p72syk (20, 21). The probe sequences were 5'-ACATTCACCGGGACCTGCGAGC-3' and 5'-CAGCTGGACAACCCGTACATCGTGC-3' for p53/56lyn and p72syk, respectively. In both instances the 5'-reporter dye was FAM and the 3'-quencher dye was TAMRA.

Quantification of mRNA Using Real Time PCR

The Real Time PCR method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System (Perkin-Elmer 7700) (22). With this instrumentation, seven measurements of fluorescence per well per PCR cycle are made during a 40-cycle amplification, and the point at which fluorescence exceeds a predetermined threshold (occurring within the linear region of the amplification curve) determines a cycle number (so-called CT) for the sample. Because multiple measurements are made during each cycle, the analytical software produces fractional cycle numbers for each sample. Using this system we determined the mRNA levels for p72syk and p53/56lyn in human basophils from a selection of releasers and nonreleasers. In pilot experiments, serial dilutions of our syk mRNA standard as well as some donor samples were evaluated and it was determined that each unit increase in cycle number (average CT = 1.03 ± 0.04 for multiple 2-fold dilutions) was equivalent to a 2-fold decrease in mRNA for both syk and lyn. The final PCR product was run on a 2.5% agarose gel to confirm that a single product at the correct base-pair length was produced. The mRNA levels of lyn and syk of donor samples were quantified as a fraction of a positive control (obtained from a large number of basophils and stored in aliquots at 130°C) run in the same assay (i.e., sample-to-standard ratio). Pilot experiments also confirmed that the mRNA data obtained by Real Time PCR was consistent for the same sample run on at least two different occassions. In addition, we also re-collected and analyzed the mRNA from one releaser and one nonreleaser on a second occasion and found the rank order distribution to be comparable to the data obtained several months earlier for both syk and lyn.

PCR Cloning and Sequencing of p72syk

Primer sets were designed using primer express and the reported cDNA sequence for p72syk. The forward primer was 5'-ATGGCTGACAGCGCCAAC-3' (nucleotide position 163, Genbank accession no. L28824) and the reverse primer was 5'-CCACGGACAGGGCGAAG-3' (position 297). This primer set flanks the region where the guanine nucleotide insertion from a mutant syk has been reported (23). Four donors were selected, two releasers and two nonreleasers, on the basis of the Real Time PCR results so that in each group there was a high and a low syk mRNA expressor (to account for possible phenotypic differences in each group). The mRNA from these donors was then reverse transcribed using MMLV reverse transcriptase (RT) according to the manufacturer's recommendations (LTI, Gaithersburg, MD). A total of 5 µl of the reverse transcription reactions were used for PCR with TAQ Gold polymerase (PE Biosystems, Foster City, CA) using the previously described primers. For the PCR reaction, 35 cycles of a 30-s denaturation step at 94°C, an annealing step at 60°C for 45 s, and an extension for 30 s at 72°C was used. The PCR products were then subcloned into a pCRII vector using the TOPO Cloning kit from Invitrogen (Carlsbad, CA). All clones were sequenced using a fluorescene-based cycle-sequencing kit and a Perkin-Elmer 377 DNA sequencer.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Protein Expression of lyn and syk

Our results confirm the recently published observation by Kepley and Andrews (14) that the expression of p72syk protein is significantly reduced in basophils from nonreleasers compared with releasers (Figure 1A). To analyze the contents of these samples, band intensities were compared with a standard included in each blot and these data are shown as a distribution plot in Figure 1B. On this basis, there was at least a 10-fold difference in expression between the two groups. Similar results were found whether whole-cell lysates or immunoprecipitates were analyzed by Western blotting. In addition, no difference was observed whether a monoclonal or polyclonal antibody was used in the Western blotting technique (data not shown). The average histamine release to anti-IgE in our releaser group was 65 ± 16% and in our nonreleaser group 1 ± 1%. However, histamine release to fMLP was comparable between releasers and nonreleasers (34 ± 5 and 28 ± 7%, respectively).

View larger version (11K): [in this window] [in a new window]   Figure 1.   Expression of p72syk in human basophils from releasers and nonreleasers. Lysates from highly purified (> 97%) releaser and nonreleaser human basophils were analyzed by Western blotting for p72syk. (A) Expression of p72syk in human basophils from two releasers and two nonreleasers. The intensity of each band was measured by densitometric analysis and expression of p72syk was then determined as a fraction of a standard run in the same gel. (B) Relative expression of p72syk as a fraction of the positive control in human basophils from releasers (filled circles) (n = 6) and nonreleasers (open circles) (n = 3).

The studies by Kepley and Andrews suggested that expression of lyn kinase might also be lower in nonreleasers (14). As Figure 2A shows, the expression of lyn protein was significantly reduced in nonreleasers compared with releasers. As for syk, we compared the band intensities with a standard included in each blot and the quantitative analysis is shown in Figure 2B. On this basis, there was at least a 10-fold difference in expression. The average histamine release in our releaser group was 62 ± 16% and in our nonreleaser group 1 ± 1%. This reduced expression of syk and lyn appears to occur only in the human basophil; the mononuclear cells obtained from the initial Percoll step gradient expressed nearly equivalent levels of these two kinases in the releaser and nonreleaser groups (data not shown). To determine whether the reduced protein level of syk and lyn in nonreleasing human basophils was a common occurrence with all signaling molecules associated with the high-affinity IgE receptor, we also examined the protein expression levels of the tyrosine kinase Btk. In many cell types, Btk has been shown to play an important role in the regulation of the sustained calcium response initiated upon receptor crosslinking (24, 25). In the human basophil Btk was strongly detected; however, its expression level was found to be similar in both releasing and nonreleasing basophils (see Figure 3).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Expression of p53/56lyn in human basophils from releasers and nonreleasers. Lysates from highly purified (> 97%) releaser and nonreleaser human basophils were analyzed by Western blotting for p53/56lyn. (A) Expression of p53/56lyn in human basophils from three releasers and one nonreleaser. The intensity of each band was measured by densitometric analysis and expression of p53/56lyn was then determined as a fraction of a standard run in the same gel. (B) Relative expression of p53/56lyn as a fraction of the positive control in human basophils from releasers (filled circles) (n = 6) and nonreleasers (open circles) (n = 2).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Expression of Btk in human basophils from releasers and nonreleasers. Lysates from highly purified (> 97%) releaser and nonreleaser human basophils were analyzed by Western blotting for Btk. (A) Expression of Btk in human basophils from two releasers and two nonreleasers. The intensity of each band was measured by densitometric analysis and expression of Btk was then determined as a fraction of a standard run in the same gel. (B) Relative expression of Btk as a fraction of the positive control in human basophils from releasers ( filled circles) (n = 4) and nonreleasers (open circles) (n = 4).

The wide distribution in syk protein expression and histamine release across these two donor groups suggested that these two variables might be correlated. Figure 4A shows a correlation between syk expression and histamine release obtained with an optimal concentration of anti-IgE antibody (Spearman rank correlation coefficient [Rs] = 0.98, P = 0.006). A similar correlation was also observed between lyn kinase expression and histamine release (Rs = 0.93, P = 0.01) (see Figure 4B).

View larger version (11K): [in this window] [in a new window]   Figure 4.   Correlation between p72syk or p53/56lyn expression and histamine release from human basophils. Lysates from highly purified (> 97%) human basophils were analyzed for the expression of either p72syk or p53/56lyn by SDS-PAGE and Western blotting. The intensity of each band was measured by densitometric analysis and the expression of p72syk or p53/56lyn was then determined as a fraction of a standard run in the same gel. In the same cells, the amount of histamine release to an optimal concentration of anti IgE antibody was also determined. (A) The correlation between p72syk expression (as a fraction of a standard) and histamine release is shown in human basophils from nine individual donors. Rs = 0.98, P = 0.006. (B) The correlation between p53/56lyn expression (as a fraction of a standard) and histamine release is shown in human basophils from nine individual donors. Rs = 0.93, P = 0.01.

mRNA Expression of lyn and syk

We next examined the expression of mRNA levels for both lyn and syk kinase in these two groups. Using Real Time PCR, we found no difference in the expression levels of mRNA for lyn and syk (Figure 5). The results are expressed as a fraction of a standard run in each assay. Analysis of the mononuclear cells from the same preparations also showed approximately equal levels of mRNA for lyn and syk in both groups. However, the amount of mRNA in the mononuclear cells was approximately 10-fold greater than in the basophil from the same donor.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Expression of p72syk and p53/56lyn mRNA levels in human basophils from releasers and nonreleasers. Human basophils were purified (> 98%) from either releasers (filled circles) or nonreleasers (open circles) and the the expression of p72syk and p53/ 56lyn mRNA levels determined using Real Time PCR. mRNA expression levels for (A) p72syk and (B) p53/56lyn in human basophils from releasers and nonreleasers. (The initials of the donors used in this experiment are shown next to each data point.)

Partial Sequencing of the syk mRNA

These results demonstrate that the absence of detectable syk and lyn protein cannot be explained by alterations in the expression of mRNA for syk or lyn. In addition, there was normal protein expression of lyn and syk in the contaminating cells associated with these preparations, so it is unlikely that a mutation in the syk gene could be responsible for these results. We therefore examined whether the differential expression in syk may be due to a syk mutation that could consequently result in a post-translational modification such as premature degradation of the syk protein. In the Jurket E6-1 cell line it has previously been shown that a mutation consisting of a single nucleotide insertion can exist in the syk sequence which leads to a premature stop codon (23). The resulting transcripts are capable of encoding only the first 33 amino acids of the 630-amino acid wild-type syk.

mRNA was isolated from the basophils of two nonreleasing and two releasing donors. RT-PCR was performed to determine the expression of syk mRNAs in these donors using forward and reverse primers that annealed around the appropriate region (see MATERIALS AND METHODS). Our initial results reconfirmed our Real Time PCR results and demonstrated that mRNA for syk was present in basophils from both releasing and nonreleasing donors (data not shown) as well as an appropriately sized product. The amplified cDNAs were then isolated, cloned, and sequenced from each of the four donors (two releasers and two nonreleasers). Our results indicate that the partial cDNAs for each of the donors were found to be identical to each other and to the wild-type syk (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been recognized for a number of years now that although basophil histamine release varies among donors there are some donors whose basophils completely fail to release histamine in response to anti-IgE stimulation (26). In the present study we have presented data showing that these nonreleasing donors have at least a 10-fold lower protein expression of p72syk. Interestingly, these nonreleasing donors also had a lower protein expression of the tyrosine kinase p53/56lyn. It seems unlikely that these differences could result from unforeseen technical issues inasmuch as expression levels of Btk, another putative tyrosine kinase in the IgE-mediated pathway, were found to be normal. Although this data may suggest that a common element may regulate the expression of these two signaling molecules, we are uncertain whether the reduced expression of both of these kinases contribute to the nonreleaser phenotype. Deficiency of syk alone might account for the nonreleaser phenotype. Previous studies using syk-deficient mast cells have demonstrated that these cells are unable to degranulate, synthesize leukotrienes, and secrete cytokines when stimulated through the high-affinity IgE receptor (27, 28), an effect which cannot be overcome by the transfection of another syk family kinase, Zap-70 (29). In contrast, bone marrow-derived mast cells from lyn knockout mice can produce a normal secretory response after IgE-receptor crosslinking due to the compensatory activation of other src family kinases (30). In the human basophil, it is unclear what other, if any, src family kinases are expressed in these cells in addition to p53/56lyn. Our own unpublished observations indicate that they do not express p60^c-src or p62^c-yes; however, there are many other src family kinases which may be present and able to compensate for this deficiency in lyn kinase. Nevertheless, having two early signaling components depressed could result in the especially poor release that characterizes these donors. Further studies will have to be done to address whether the reduced expression of lyn contributes to the nonreleaser phenotype.

We next looked at the mRNA levels of lyn and syk to determine whether their reduced protein expression in the nonreleaser phenotype resulted from differences in transcriptional regulation. We found that lyn and syk mRNA levels were comparable for both groups of donors, suggesting that differences in gene transcription or mRNA stability are not responsible for differences in protein expression. However, a reduction in syk and lyn protein was not observed in other cell types, including monocytes and lymphocytes, suggesting that this phenomenon is unique to the human basophil in these donors and is therefore unlikely to be due to a mutation in the syk or lyn gene. Further, it seems unlikely that a mutation could exist in both lyn and syk to specifically suppress their expression in human basophils. Nevertheless, although unlikely, it remains possible that there is a mutation in these proteins leading perhaps to the premature degradation of the syk or lyn protein in the context of the basophil intracellular environment.

To date, the only known mutation in the syk gene which is known to have functional consequences has been demonstrated in the Jurket E6-1 T-cell line (23). In these studies, sequencing of the syk mRNA revealed that the Jurket E6-1 T-cell line had a nucleotide insertion at position 92 in the syk open reading frame. The resulting frame shift allowed for the alternative usage of two codons before directing the premature termination of the open reading frame at position 109. This severely truncated gene product was then not recognized by conventional Western blotting techniques, though mRNA levels were normal in these cells (23). We found no indication that a similar frame-shift mutation occurs in the nonreleasing human basophil. This does not, however, exclude the possibility that a mutation exists in a part of the syk protein not sequenced in this study.

Following the above chain of logic, we tentatively conclude that there is an alteration in either the translational or post-translational processing of these two proteins. Studies are still ongoing to determine what alteration in these nonreleasing human basophils influences the translational or post-translational processing of syk and lyn kinase. We have also not ruled out the possibility that this may be a post-translational effect in these cells which is influenced by the intracellular environment of the basophil. It has previously been shown that various tyrosine kinases are susceptable to proteolytic cleavage, including p72 and pp60v-src (31, 32). However, no direct evidence has been found for the cleavage of p72syk or p53/56lyn, though they do share structural features similar to p72 and p60src.

We were intrigued with the observation that there was a wide distribution in the mRNA for both lyn and syk among donor basophils. Additional experiments confirmed that this distribution was constant over various Real Time PCR assays and across different mRNA samples for the same donors over a 3-mo period (see MATERIALS AND METHODS). However, whether this variability in mRNA for syk and lyn has any functional relevance will need to be addressed in future studies. A significant relationship was also found between syk (P = 0.006) and lyn (P = 0.01) protein expression and histamine release. This result was somewhat unexpected because previous studies suggested that expression levels of either of these proteins might influence the sensitivity of the cell rather than the maximal release obtainable (1, 33).

In summary, we found that nonreleasing human basophils have at least 10-fold less protein expression of the tyrosine kinases lyn and syk compared with releasing basophils, but normal levels of the tyrosine kinase Btk. In addition, the mRNA levels for lyn and syk are similar for the two groups. We therefore conclude that nonreleasing human basophils have a translational or post-translational alteration in their expression of the tyrosine kinases lyn and syk.