Table 1.
Patient characteristics*.
Fig 1.
MAPKs phosphorylation in freshly isolated PBMC of SS asthmatics and SR asthmatics as assessed by Western blot.
(A) Cell lysates were prepared from the PBMC of nine subjects with SS asthma and nine subjects with SR asthma. 50 μg of protein from each patient PBMC cell extract were run on a 4–20% gradient gel and blots were probed with antibodies against phosphorylated forms of p38, ERK and JNK. Membranes were stripped and re-probed with antibodies against total p38, ERK and JNK, respectively. Hsp90 detection was used to monitor protein loading. (B) Densitometry values of phosphorylated form of p38 to total p38 in PBMC of SR and SS asthmatics.
Fig 2.
Flow cytometry assessment of MAPK phosphorylation in whole blood samples of SR and SS asthma patients.
Whole blood aliquots were stained with either anti-CD3, CD4, CD8 antibody cocktail or CD14, CD16, CD20 antibody cocktail plus one of the phospho-MAPK antibodies (i.e. p-p38, p-ERK, p-JNK) or corresponding IgG isotype control antibodies (to detect nonspecific antibody binding). (A) Phosphorylation of p38 in CD14+ monocytes of subjects with SR asthma vs. SS asthma (MFI). (B) Phosphorylation of ERK in CD14+ cells of subjects with SR asthma vs. SS asthma (MFI).
Table 2.
MFI of phosphorylated MAPK staining in various cell types in the blood samples of asthmatics.
Fig 3.
Activation of the p38 pathway in the cells of SR asthmatics.
(A) Significantly elevated p-MSK1 levels in lysates from PBMC of SR as compared to SS asthma patients. Representative Western blot results of p-MSK1 and MSK1 in PBMC are shown. PBMC protein lysates prepared to assess p38 phosphorylation (Fig 1) were examined for p-MSK1 expression. (B) Densitometric values of phosphorylated form of MSK1 to total MSK1 in PBMC of SR and SS asthmatics.
Fig 4.
Inhibition of p38 MAPK activation using p38 inhibitor SB203580 results in increased DEX suppression LPS-induced IL-8 mRNA production.
P38 MAPK inhibition by p38 inhibitor SB203580 (SB), but not ERK inhibitor, PD98059 (PD) results in selective inhibition of downstream kinase activation (MSK1 phosphorylation) in the cells of SR asthmatics as shown by Western blot (A, B). After preincubation of PBMC from SR asthmatics with a selective p38 inhibitor the cells became steroid sensitive, i.e. greater suppression of LPS induced IL-8 mRNA by DEX, as shown by RT-PCR (C).