Mechanisms of asthma and allergic inflammation
Relationship between spleen tyrosine kinase and phosphatidylinositol 5′ phosphatase expression and secretion from human basophils in the general population

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Background

Previous studies have suggested that expression levels of spleen tyrosine kinase (syk) or phosphatidylinositol 5′ phosphatase (SHIP) may explain certain extreme human basophil phenotypes.

Objective

This study is designed to explore whether variability in syk and SHIP expression levels in the general population, alone or in concert, can account for the variability in basophil function.

Methods

A survey of maximum responsiveness to IgE-mediated stimulation, sensitivity, and expression levels of 6 early signaling elements was performed on 36 subjects' basophils.

Results

Of the 6 signaling elements, only syk and SHIP showed a correlation with maximum histamine release or cellular sensitivity. In a multiple regression, syk and SHIP together could account for 67% of population variance, although most of the variance was explained by syk expression. The pattern of expression variance syk>>SHIP1>SHIP2≈lyn≈p85≈cbl suggested a process that primarily modulated syk levels. IL-3 is known to modulate syk levels, but we found that a 3-day incubation with IL-3 resulted in increased expression of other signaling elements to a greater degree: cbl>SHIP1>SHIP2≈lyn≈p85≥syk, opposite the pattern in the population survey. In contrast, 18-hour stimulation with anti-IgE antibody led to marked downregulation of syk expression, modest downregulation of FcɛRI expression, weak downregulation of lyn expression, and no effect on 23 other signaling elements.

Conclusion

Unlike studies in mice, we conclude that expression of syk is a good preconditioning predictor of basophil function in the general population.

Clinical implications

The finding that expression of syk levels may strongly influence functional responses of basophils suggests a mechanism underlying the severity of atopic diseases.

Section snippets

Materials and buffers

The Online Repository (www.jacionline.org) provides the description of standard materials, buffers, and antibodies used for these studies. Briefly, various piperazine-N,N′-bis(2-ethanesulfonic acid) buffers, as described previously, were used for short periods of stimulation, and supplemented RPMI-1640 was used for overnight incubations.17 RPMI-1640 for overnight cultures contained 8 μg/mL gentamycin and 300 μg/mL human serum albumin, supplemented to 1 mmol/L final Ca++ concentration. The syk

Results

To assess the variation in interassay sample error, whole cell lysates from 9 different subjects were run in multiple Western blots. From these measurements, we found approximately ± 24% (error relative to measured amount) variation for the syk kinase determination and ± 11% variation for SHIP1. We also examined expression levels in basophils of 9 subjects with a second purification and Western analysis; expression levels showed a relative variation of 21% and 35% for syk and SHIP1,

Discussion

Previous studies on the relationship between signal element expression and basophil function examined extreme differences in function. In addition, the codependent relationship between signal element expression and function has not been examined. The current studies provide a broader context for signal elements expression. There are certainly many signaling elements absolutely required for mediator release, but it appears that syk expression is a focus of natural variation. Indeed, the

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  • Cited by (0)

    Supported by National Institutes of Health grant AI20253.

    Disclosure of potential conflict of interest: The author has declared that he has no conflict of interest.

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