Zymosan, SU6656 and wortmannin, were from Sigma Chemical Co. (St Louis, MO, USA). Heat-killed S. aureus was kindly provided by Dr Lars Björck, BMC, Lund University. PP2 was from Biomol (Plymouth Meeting, PA, USA), while SP600125 was from Tocris Cockson (Northpoint, UK). LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA). [³H]Arachidonic acid (196 Ci/mmol) was from Amersham international (Little Chalfont, UK). Antibodies against p-JNK, PLC-γ2, ERK-2 and phosphotyrosine as well as HRP conjugated secondary antibodies, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-ERK, p-p38 and pAkt (Thr308) were from New England Biolabs (Beverly, MA, USA). Antibodies against pAkt (Ser473) and pBtk (Tyr223) were from Cell Signaling Technology (Beverly, MA, USA). The Mnk-1 inhibitor CGP057380 was a kind gift from Dr Hermann Gram, Novartis Pharma AG, Basel, Switzerland. Zymosan and bacteria were dispersed in PBS. Zymosan was used at 0,2 mg/ml and bacteria at a concentration of 2,5*10^7/ml. The inhibitors LFM-A13, PP2, CGP057380, SU6656, SKI-1 and wortmannin were dissolved in dimethyl sulphoxide (DMSO) and added to cells resulting in a final concentration of DMSO of less than 0.3%.

Peritoneal macrophages were isolated from female outbred NMRI mice by adherence in a humidified atmosphere of 5% CO₂ in air at 37°C. Non-adherent cells were removed after 2 h and the rest of the cells were incubated in medium 199 supplemented with 1% fetal bovine serum for 18–20 h. During this incubation cells were labelled with [³H]arachidonic acid in some experiments.

Cells labelled with 0,5 μCi [³H]arachidonic acid/well for 18–20 h, were washed three times with PBS, and stimulated in serum-free medium. At the end of the experiment the medium was collected and the cells scraped off the dishes in 0,1% Triton X-100 in water. The collected medium was centrifuged and the arachidonate released from cellular phospholipids was determined by liquid scintillation counting. The release into the collected medium is expressed as a percentage of total recovered radioactivity in cells and medium.

Cells were cultured in 10 cm² dishes and stimulated in a serum-free medium. When inhibitors were used, they were added 15 min. before stimulus. After stimulation the cells were scraped off the dishes in 110 μl sample buffer and equal amounts of cell lysate were subjected to SDS-PAGE. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes which were blocked in 3% or 5% non-fat milk/gelatin for 1 h followed by incubation with different antibodies. Bound antibodies were detected with secondary horseradish peroxidase-labeled antibodies and enhanced chemiluminescence using LAS 1000 Plus (Fuji Film, Stockholm, Sweden). To assure equal loading of the gels the membranes were stripped and reprobed with ERK-2 antibody, or as indicated.

Macrophages (approx. 10⁶ cells) were stimulated in serum-free medium. When inhibitors were used, they were added 15 min. before the stimulus. At the end of the experiment cells were washed with ice-cold PBS and scraped off the dish in lysis buffer (0.1 M TrisCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % nonionic detergent (Igepal, polyoxyethylene nonylphenol), 20 mM NaF, 1 mM Na₃Vo4, 1 mM PMSF and 1 μg/ml each of pepstatin and leupeptin). After centrifugation (10⁴ × g) for 10 min at 4°C the supernatant was incubated with an antibody against PLC-γ2. The immune complexes were captured using protein A-Sepharose (50% w/v). The samples were centrifuged and the immune complexes were washed three times in lysis buffer. The immunoprecipitated proteins were dissolved in 2X sample buffer and subjected to SDS-PAGE (7% acrylamide) and analyzed by Western blotting.

Both zymosan and Gram-negative bacteria induce arachidonate release in mouse macrophages 4, zymosan being the more potent of the two classes of stimuli. SFK are important in many signalling pathways, but their role in the present process is so far unknown. To investigate this two different Src kinase inhibitors (PP2 and SKI-1) were used. Both SFK inhibitors reduced the response induced by zymosan and Gram-positive S. aureus in a concentration dependent manner (Fig. 1). The decrease at 5 μM PP2 was approximately 59% for zymosan and 84% for S. aureus. SKI-1 (5 μM) inhibited the zymosan-induced release by approximately 82% and the S. aureus-induced release by 83%.

The MAP kinases ERK and p38 are both known to contribute to the activation of cPLA₂ in response to zymosan or bacteria 4. Both zymosan and whole bacteria induce regulatory phosphorylations of these MAP kinases and we show here that pretreatment of the cells with PP2 (1–10 μM) decreased these phosphorylations in the case of not only ERK and p38, but also of JNK (Fig. 2A, B). Also SKI-1 was able to decrease the zymosan- and bacteria-induced phosphorylation of ERK (Fig. 2C).

To establish whether also JNK might play a role in the activation of cPLA₂ SP600125, a JNK inhibitor 18 was tried. Pretreatment with SP600125 (5–40 μM) led to a concentration dependent decrease of the zymosan-induced arachidonate release. However, this was accompanied by a parallel inhibition of the phosphorylation of the MAP kinases ERK, p38 and JNK as detected by antibodies against the phosphorylated MAP kinases (not shown). Thus, under our conditions SP600125 may also act as an inhibitor upstream of several MAP kinases.

It has been suggested that the kinase Mnk-1, downstream of both ERK and p38, might transmit the signal to some of the phosphorylations necessary for the activation of cPLA₂ 5. Treatment of cells with the Mnk inhibitor CGP 57380 (20 and 40μM) resulted in partial inhibition (41 and 50%, respectively) of the arachidonate release in response to zymosan (Table 1).

We have previously shown that PI3K has an important role in the zymosan and bacteria induced signalling to cPLA₂ activation and arachidonate release 19. PI3K can cause activation of the serine/threonine protein kinase Akt. The activation is dependent on phosphorylation of two sites, one in the activation loop of the kinase core (Thr 308) and one near the carboxy terminus (Ser 473). Zymosan beads, in contrast to S. aureus, induced Akt phosphorylation at both Ser 473 and Thr 308 in macrophages, as detected by phosphospecific Akt antibodies (Fig. 3A). The phosphorylation of both sites appeared to depend on SFK activation and PI3K. This can be seen as decreases in these phosphorylations after inhibition with either PP2 or wortmannin (Fig. 3B).

PLCγ2 is considered to be activated by tyrosine phosphorylation and/or by the product of PI3K, but both PI3K-dependent and -independent pathways leading to activation have been demonstrated. The role of Src kinases in zymosan-induced PLCγ2 activation was investigated by immunoprecipitation with a PLCγ2 specific antibody. A prominent tyrosine phosphorylation was induced by zymosan, but was not detected in cells exposed to S. aureus (Fig. 4A). Pretreatment of the cells with PP2 (Fig. 4B) or SKI-1 (Fig. 4C) clearly inhibited the zymosan-induced tyrosine phosphorylation of PLCγ2. In agreement with previous findings 3, inhibition of PI3K with wortmannin did not cause such inhibition (Fig. 4B).

Btk is a member of the Tec family of cytoplasmic tyrosine kinases. Most studies on Btk have been conducted with B-lymphocytes, where cell activation leads to membrane translocation of Btk and phosphorylation of two sites (Tyr 551 and Tyr 223). Tyr 551 is situated in the activation loop and its phosphorylation may be initiated by SFK, leading to autophosphorylation of Tyr 223, which appears necessary for full activation 2021. Detection of Btk activation by phosphospecific (Tyr 223) antibody showed that a low level of phosphorylation on Tyr 223 was present in control cells (Fig. 5A). Furthermore, zymosan and S. aureus but not LPS or peptidoglycan induced phosphorylation of Btk in macrophages leading to enhanced immunostaining of the band detectable in control cells as well as appearance of an additional band, presumably due to additional phosphorylation(s) causing gel-shift (Fig. 5A). Pretreatment with the PI3K inhibitor wortmannin or the SFK inhibitors PP2 and SKI-1 caused total, or pronounced, inhibition of zymosan-induced phosphorylation of Btk (Fig. 5B). A Src kinase inhibitor with a different specificity (SU6656;22) was much less inhibitory (Fig. 5B). A different inhibitor profile was observed when S. aureus was used as stimulus (Fig. 5C). Btk phosphorylation (on Tyr223) was then insensitive to wortmannin, but sensitive to all three SFK inhibitors, including SU6656. These results, together with differences in the phosphorylation of Akt (Fig. 3A) and PLCγ2 (Fig. 4A) argue for differential engagement of individual SFK members as well as of PI3K in the response to zymosan and S. aureus.

Interestingly, an inhibitor of Btk (LFM-A13) caused a decrease in the release of arachidonate induced by both zymosan and S. aureus (Fig. 6A). The decrease was prominent at 10 μM LFM-A13 with little further change at higher concentrations (approx. 65% inhibition for zymosan and ≥ 80% for S .aureus at 50 μM LFM-A13). However, in contrast to the effect of SFK inhibitors described above, the inhibition of Btk did not affect the zymosan- and bacteria-induced phosphorylation of ERK and p38 (Fig. 6C), neither did it affect the zymosan-induced phosphorylation of PLCγ2 (Fig. 6B). A partial inhibition of the phosphorylation of Akt induced by zymosan was observed (Fig. 6D), but only at higher concentrations than required for inhibition of arachidonate release (Fig. 6A).

These data indicate that Btk has a regulatory role for arachidonate release, is acting downstream of inhibitor-sensitive SFK, but not involved in signalling to MAP kinase activation.