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(Journal of Leukocyte Biology. 2000;68:575-582.)
© 2000 by Society for Leukocyte Biology

Differential localization of protein kinase C isotypes in equine eosinophils and neutrophils

E. C. Greenaway, F. M. Cunningham* and N. T. Goode

Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London, NW1 OTU, UK; and
* Hawkshead Campus, Hawkshead Lane, Hatfield, Herts, AL9 7TA, UK

Correspondence: N. T. Goode, Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London, NW1 OTU, UK. E-mail: ngoode{at}rvc.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC {alpha}, ß, {delta}, {varepsilon}, {iota}, and {zeta}. However, unlike the equine neutrophil, the majority of the PKC was detected in the particulate fraction of the cell. Despite this unusual location, the PKC in equine eosinophils was activatable, suggesting that it is functionally competent. The regulatory role of PKC in equine eosinophils may reflect the association of activity with the particulate fraction and the profile of isotype expression.

Key Words: horse granulocytes • PKC isotypes • PKC inhibitors • superoxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils play a prominent role in host defense against parasitic infestation in the horse. However, these cells have been implicated also in the pathogenesis of allergic diseases, such as the skin condition sweet itch [1 ]. Equine eosinophils accumulate in large numbers in lesional skin, and inappropriate activation of these cells will lead to the release of a range of enzymes, basic proteins, mediators, and free radicals that have cytotoxic or inflammatory properties [2 3 4 5 6 7 ]. A number of mediators, which can cause the adherence, migration, or activation of equine eosinophils, have been identified. These include substance P, platelet activating factor (PAF), histamine, leukotriene (LT) B4, and the complement fragment C5a; evidence that PAF and histamine are released following antigen challenge in sweet itch has been obtained [7 8 9 10 11 12 ]. At present, however, little is known about the downstream signaling events initiated by these mediators binding to their cell-surface receptors or the regulation of the functional responses of equine eosinophils. Understanding the regulatory mechanisms in these cells could reveal potential targets upon which drugs may act to decrease eosinophil accumulation or activation in allergic diseases.

Protein kinase C (PKC) is a family of serine/threonine protein kinases consisting of 12 different isotypes [13 ]. Studies of eosinophil function in other species using PKC inhibitors suggest that PKC is involved in a range of eosinophil functions in response to different stimuli. Thus, the respiratory burst induced by vascular cell adhesion molecule 1 (VCAM-1), granulocyte-macrophage colony-stimulating factor, PAF, or CD11b/CD18 in human cells is reduced in the presence of such inhibitors, as is activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in elicited guinea pig eosinophils in response to PAF or LTB4 [14 15 16 17 ]. Human eosinophil apoptosis is also reported to be decreased by PKC inhibitors, suggesting PKC may be important for cell survival [18 ]. However, it should be noted that not all the functional responses that have been studied to date are PKC-dependent. For example, although CD11b/CD18-induced superoxide production is PKC-dependent, adherence in response to this stimulus is not [17 ]. PAF-induced, but not interleukin-5-induced, chemotaxis is also PKC-independent [19 ]. Moreover, PAF-induced eicosanoid production by human eosinophils is reported to be enhanced in the presence of PKC inhibitors [20 ], suggesting PKC-mediated suppression of this response.

Phorbol 12-myristate 13-acetate (PMA), which produces its effects by activating PKC [21 , 22 ], has been shown to increase the adherence and superoxide production of equine eosinophils [7 , 10 ]. Moreover, expansion of fusion pores in the membrane of these cells, which precedes exocytosis, is reduced in the presence of PKC inhibitors [23 ]. These findings, taken together with data implicating PKC in the regulation of functional responses in eosinophils from other species, suggest that PKC could also be important in regulating equine eosinophil function. The aim of the present study was, therefore, to establish which PKC isotypes are present in equine eosinophils so that, in future studies, the role of individual PKCs in regulating cell adherence, migration, and activation can be investigated. Thus, PKC isotype-selective therapies may be identified as appropriate to evaluate in allergic diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Hanks’ balanced salt solution (HBSS) was obtained from Life Technologies (Paisley, Scotland). Percoll was from Pharmacia (Milton Keynes, UK). Okadaic acid, N,N,N',N-tetra kis(2-Pyridymethyl)-ethylene diamine (TPEN), staurosporine, and GF109203X (bisindolymaleimide I) were from Calbiochem (Nottingham, UK). The monoclonal antibodies (mAbs) to PKC ß, {theta}, {lambda}, µ, and {iota} (raised against human PKCs) were from Signal Transduction (Exeter, UK). The polyclonal antibodies and epitope peptides to PKC {alpha} (raised against human PKCs) and PKC {delta}, {varepsilon}, and {zeta} (raised against rat PKCs) were from Santa Cruz Biotechnology (Wembley, UK). The polyclonal antibodies and epitope peptides to PKC ßI, ßII (raised against bovine PKCs), and PKC {eta} (raised against mouse PKC), and the mAb to PKC {gamma} (raised against human PKC) were kindly donated by Professor Peter Parker (Imperial Cancer Research Fund, London) [24 , 25 ]. The {delta} pseudosubstrate-site peptide was kindly donated by Dr. C. Pears (Biochemistry Department, University of Oxford, UK). All other chemicals were obtained from Sigma Chemical Company (Poole, UK) or BDH Laboratory Supplies (Poole, UK).

Purification of equine eosinophils and neutrophils
Venous blood samples (300 ml) from healthy New Forest geldings were collected into ethylenediaminetetraacetate (EDTA; final concentration, 10 mM), and eosinophils and neutrophils were purified as previously described [7 ]. Briefly, blood was allowed to sediment at room temperature for 20 min before the leukocyte rich plasma (LRP) was collected and centrifuged (200 g, 4°C, 5 min). The majority of the leukocyte poor plasma (LPP) was removed, and the cell pellet resuspended in 7.5 ml LPP. The cell suspension was then layered onto a 70:85:100% (5 ml each) Percoll density gradient and centrifuged (400 g, 4°C, 15 min). Eosinophils were harvested from the 85:100% interface and from within the 100% layer. Neutrophils were harvested from the 70:85% interface. The cells were washed in 20 ml 1 x Ca2+- and Mg2+-free HBSS and centrifuged (200 g, 4°C, 5 min). The remaining erythrocytes were lysed by addition of 10 ml of 34 mM NaCl followed by 10 ml of 270 mM NaCl, centrifuged (200 g, 4°C, 5 min), and washed as before. Eosinophils were used immediately in the superoxide assay or frozen at -80°C as a dry pellet, containing 2 x 107 cells pooled from four horses, until required. Neutrophils were frozen as dry pellets containing 2 x 107 cells pooled from the same four horses from which eosinophils had been prepared.

PMA-induced superoxide anion generation
Superoxide anion generation was measured by the superoxide dismutase inhibitable reduction of cytochrome C (cyt C) as previously described [7 ]. Equine eosinophils were suspended at a final concentration of 0.25 x 106 cells/ml in 1 x HBSS containing the following: 2.5 mg/ml cyt C, 4 mM NaHCO3, 10 mM HEPES, 0.125% (w/v) bovine serum albumin (BSA), pH 7.4. Cell purity and viability were assessed by Diff Quick and Trypan Blue exclusion, respectively, and were found to be >96%. Eosinophils were incubated with PMA (1 pM-10 nM) for 30 min at 37°C and centrifuged (1400 g, 20°C, 10 min), and 250 µl supernatant was transferred to wells of a 96-well microtitre plate. The absorbance was then measured at 550 nm using a Spectromax 250 plate reader (Molecular Devices, Crawley, UK). Superoxide production was calculated as nmol cyt C/106 cells. Each concentration of PMA was assayed in triplicate, and mean values were then calculated using eosinophils from three ponies. In studies of PKC inhibition, the cells were incubated with staurosporine or bisindolymaleimide I (0.1 nM-1 µM) for 5 min at 37°C prior to the addition of 1 nM PMA.

Identification of PKC isotypes
Preparation of rat and equine brain
Rat brain or equine cerebrum (0.5 g) was suspended in 2 ml homogenizing buffer [20 mM Tris, pH 7.5, 10 mM EGTA, pH 8, 1 mM EDTA, pH 8, 250 mM sucrose, 0.2% (w/v) Triton X-100, 0.1% (w/v) ß-mercaptoethanol, 20 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.02% (w/v) leupeptin] and homogenized using a Dounce homogenizer. The samples were incubated on ice for 1 h, centrifuged (100,000 g, 4°C, 1 h), and the supernatant was then mixed with equal volumes of 2 x sodium dodecyl sulfate (SDS) sample buffer [26 ]. Following incubation at 95°C for 5 min, the samples were cleared of debris by centrifugation at 13,000 rpm for 5 min in a microcentrifuge and stored at -20°C until analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) [27 ].

Preparation of equine eosinophils and neutrophils
Eosinophils and neutrophils (2x107 cells) were resuspended in 1 ml homogenizing buffer (as above) without Triton X-100, to which 10 mM sodium fluoride and 0.02% (w/v) aprotinin had been added. The cells were sonicated, incubated on ice for 1 h, and then centrifuged (100,000 g, 4°C, 1 h). The supernatant (cytosolic fraction) was removed and mixed with one-tenth vol 10 x SDS sample buffer [28 ]. The pellet was resuspended in homogenizing buffer containing 1% Triton X-100, 10 mM sodium fluoride, and 0.02% (w/v) aprotinin; incubated for 5 min; and then centrifuged as before. The supernatant (membrane fraction) and pellet (detergent-insoluble fraction) were mixed with 2 x SDS sample buffer. Each fraction was then incubated at 95°C for 5 min and cleared of debris as described above. The protein content in each fraction was determined (Lowry kit, Sigma Chemical Company).

For the studies of the effects of okadaic acid and TPEN on PKC localization, eosinophils were purified as described above except that okadaic acid (1 µM) [29 ] or TPEN (50 µM) [30 ] was added to the LRP at the start of the cell separation and was incubated for 15 min at 37°C before loading onto Percoll gradients. Okadaic acid (1 µM) or TPEN (50 µM) was also added to the medium after each wash.

Western blotting
SDS-PAGE was carried out as previously described [27 ]. Briefly, protein samples (35 µg) were loaded onto an 8% polyacrylamide gel. After electrophoresis at 30 mA for 2 h, the resolved proteins were transferred to nitrocellulose membranes using a semi-dry electroblotter, and then blocked in 5% (w/r) skimmed milk powder (Marvel) in Tris-buffered saline (TBS), pH 7.5, for 12 h. The membranes were incubated with the PKC isotype-specific antibodies overnight, washed three times for 10 min in TBS containing 0.05% (w/v) Tween 20, and then incubated with anti-rabbit or mouse horseradish peroxidase-conjugated antibodies for 1.5 h, as appropriate. After three further washes, visualization was achieved using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Bucks, UK) and radiography. Antibody specificity was confirmed using antibody-specific epitope peptides that were preincubated with the primary antibody (4°C, 2 h) before addition to the membranes.

Subcellular fractionation
Detailed fractionation was performed from the method of Roodyn [31 ]. Briefly, freshly prepared eosinophils (2x107) were sonicated and incubated on ice for 1 h in 1 ml of homogenizing buffer. The nuclear fraction was then pelleted by centrifugation at 1000 g for 2 min at 4°C and then incubated for 5 min in homogenizing buffer containing 1% Triton X-100. This sample was then separated into the insoluble (containing nuclear matrix) and soluble (containing nuclear membranes) nuclear fractions by centrifugation at 1000 g for 2 min at 4°C. The supernatant obtained from the first centrifugation, comprising all material except the nucleus, was divided into four further fractions. Firstly, the pellet generated by centrifugation at 20,000 g for 20 min at 4°C contained the lysosomes, peroxisomes, and mitochondria. The corresponding supernatant was centrifuged at 100,000 g for 1 h at 4°C. The resulting supernatant comprised the cytosolic fraction, and the pellet was divided further by resuspension in homogenizing buffer containing 1% Triton X-100, incubation on ice for 5 min, and centrifugation at 100,000 g for 1 h at 4°C. The resulting supernatant contained detergent-soluble membraneous structures (small vesicles, plasma membrane, and microsomes), and the pellet contained the cytoskeleton. All six fractions were placed in SDS sample buffer to the same final volume and boiled for 5 min before being analyzed by Western blotting.

PKC activity
Freshly prepared eosinophils (2x107) were sonicated and incubated on ice for 1 h in 1 ml homogenizing buffer containing 1% Triton X-100, 10 mM sodium fluoride, and 0.02% (w/v) aprotinin and then separated into detergent-soluble and detergent-insoluble fractions by centrifugation (100,000 g, 4°C, 1 h). The pellet (detergent-insoluble) was resuspended in 0.25 ml buffer. The fractions were then assayed for PKC activity, by an in vitro kinase assay [25 ] using the conventional PKC substrate histone IIIS and {delta} pseudosubstrate-site peptide, which is a substrate for the conventional and novel PKC isotypes. Briefly, 10 µl of substrates histone IIIS (5 mg/ml) or a peptide based on the pseudosubstrate site of PKC {delta} (10 µg/ml) in 50 mM Tris, pH 7.5, 0.5 mM EDTA was added to tubes containing 10 µl cell extract or buffer as control. MgCl2 (10 µl of 50 mM) and 10 µl of 20 mM HEPES, 1% (w/v) Triton X-100, were then added to the samples that were being tested in the absence of cofactors. Ten µl of 50 mm MgCl2, 3 mm CaCl2, and 10 µl of 20 mM HEPES, 1% (w/v) Triton X-100, containing 5 mg/ml phosphatidylserine and 1 µg/ml PMA, were added to the remaining tubes. Finally, the assay was initiated by adding 5 µl of 1 mM [32P-{gamma}] adenosine 5'-triphosphate (ATP; specific activity, ~1.12 MBq per pmole) to each tube and incubating at 30°C for 7.5 min. Each cell fraction was assayed in triplicate using each substrate in the presence and absence of the PKC cofactor, phosphatidylserine, and PMA. The assay was terminated by placing 30 µl aliquots of the assay mixtures onto 2 cm2 pieces of P81 phosphocellulose paper, which was then washed three times (10 min each) in 70 mM phosphoric acid with gentle stirring. The paper squares were then transferred to vials, and the bound radioactivity was assessed by Cerenkov counting. The activity was expressed as pmole phosphate transferred from ATP to substrate per min. The amount of PKC activity can be determined by subtracting cofactor-independent activity from cofactor-dependent activity.

Statistical analysis
The inhibitory effects of phorbol ester, staurosporine, and bisindolymaleimide I on superoxide production were analyzed for significance using repeated measures of one-way analysis of variance followed by Dunnett’s test. Values of p < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of staurosporine and bisindolymaleimide I on PMA-induced superoxide production
As previously described [7 ], PMA caused concentration-related superoxide production by equine eosinophils (Fig. 1a ). Staurosporine and bisindolymaleimide I inhibited 1 nM PMA-induced superoxide anion production in a concentration-dependent manner (Fig. 1b and 1c) . Complete inhibition of the response occurred using 100 nM staurosporine (p<0.02) or 1 µM bisindolymaleimide I (p<0.02). Addition of 1 µM Ro31-8220 also completely inhibited PMA-induced superoxide production (p<0.05; unpublished results).



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  		Figure 1. Effect of phorbol ester and PKC inhibitors on superoxide production in equine eosinophils. Effect of PMA on superoxide production (A). Inserts show the effects of staurosporine (B) and bisindolymaleimide I (C) on 1 nM PMA-induced superoxide production. Each point represents the mean of values obtained from three horses. Bars represent SEM. *p < 0.02.

 
Expression of PKC isotypes in equine eosinophils
A range of PKC isotypes is known to be expressed in brain tissue [26 ]. Preliminary experiments using horse brain were, therefore, carried out to establish the cross-reactivity of the PKC isotype-selective antibodies to horse PKCs. Rat brain was used as a positive control. Eight PKC isotypes were identified in equine brain; the same isotypes were present in rat brain (Fig. 2A Table 1 ). Preincubation of antibodies with epitope peptides showed that the reactivity was specific (Fig. 2B) . The molecular weight of the PKC isotypes was ~80 kDa except for PKC {varepsilon} (~90 kDa).



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  		Figure 2. Detection of PKCs in equine brain and determination of antibody specificity. A) The antibodies used were screened against rat (R) and horse (H) brain extracts, as indicated. The positive control for the PKC {lambda} antibody is a Jurkat cell line extract (J), supplied with the antibody. B) Antibody binding in horse brain extracts is competed efficiently with the epitope peptides (+). Peptides were only available with the polyclonal antibodies for PKC {alpha}, {delta}, {varepsilon}, and {zeta}. The positions of the 75 kDa molecular weight marker and the relevant PKC are indicated.

 

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  		Table 1. PKC Isotypes Expressed in Rat and Horse Brain

 
Because the PKC antibodies cross-reacted with equine tissue, equine cerebrum was used as a positive control for the studies of equine eosinophil PKC. Equine eosinophils were shown to contain conventional PKC {alpha} and ß, novel PKC {delta} and {varepsilon}, and atypical PKC {iota} and {zeta} (Fig. 3a ). PKC {gamma}, {eta}, {theta}, µ, and {lambda} were not detected (unpublished results). PKC ßI and ßII were present in equine eosinophils (unpublished results). In contrast to other cell types in which the PKC is located in the cytoplasmic fraction, the PKC isotypes in the equine eosinophil were situated predominantly in the detergent-insoluble particulate fraction. PKC {alpha}, ß, {delta}, {varepsilon}, {iota}, and {zeta} were all found in the particulate fraction. PKC ß and {delta} were also detected in the cytosol. PKC {delta} was additionally detectable in the membrane upon prolonged exposure of the gel. To ensure that the unusual location in the particulate fraction did not result from the purification and extraction protocol, we examined the location of PKC isotypes in equine neutrophils, which were prepared at the same time from the same Percoll gradients as the eosinophils. Although the same PKC isotypes ({alpha}, ß, {delta}, {varepsilon}, {iota}, and {zeta}) were found in equine neutrophils as in equine eosinophils, the isotypes were located mainly in the cytosol (Fig. 3b) . In addition, PKC {gamma} and {lambda} were detected in neutrophil cytosol, and PKC {theta} and µ were not detected (unpublished results).



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  		Figure 3. Detection and location of PKC isotypes present in equine granulocytes. Equine eosinophils (a) and equine neutrophils (b). The position of the PKC isotypes is indicated. Lanes 1 contain the cytosolic fraction of the cell, lanes 2 contain the membrane fraction of the cell, and lanes 3 contain the particulate fraction of the cell.

 
In light of the unusual location of PKC in equine eosinophils, a detailed subcellular fractionation was performed. One isotype from each class of PKC was examined ({alpha}, {varepsilon}, and {zeta}). The results for PKC {alpha} are shown (Fig. 4 ): The distribution of PKC {varepsilon} and {zeta} was similar (unpublished results). The majority of the PKC was found in the nuclear-insoluble fraction, with smaller quantities found in the nuclear-soluble fraction and the cytoskeleton.



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  		Figure 4. The subcellular location of PKC {alpha} in equine eosinophils. Equine eosinophils were fractionated and analyzed by Western blotting. Equine brain extract is used as a positive control (lane 1). The fractions are nuclear-insoluble (lane 2), nuclear-soluble (lane 3), lysosomes, peroxisomes and mitochondria (lane 4), cytosol (lane 5), cytoskeleton (lane 6), and plasma membrane, microsomes, and small vesicles (lane 7). Lanes 2–7 were loaded with extract derived from an equal number of cells. The position of PKC {alpha} is indicated.

 
Because PKC dephosphorylation and subsequent migration to a membrane location near the nucleus for degradation [32 ] could explain the unusual location of PKC in equine eosinophils, the effects of including the phosphatase inhibitor, okadaic acid, during cell separation was examined. No effect on the location of the PKC {alpha} was observed (Fig. 5A ).



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  		Figure 5. Effect of okadaic acid and TPEN on the location of PKC {alpha} in equine eosinophils. Western blot of protein extracts from equine eosinophils incubated during preparation with okadaic acid (A) or TPEN (B). Detergent-soluble fraction (lane 1); detergent-insoluble fraction (lane 2) of untreated equine eosinophils. Detergent-soluble fraction (lane 3) and detergent-insoluble fraction (lane 4) of equine eosinophils incubated with the relevant compound. The position of PKC {alpha} is indicated.

 
Equine eosinophils contain the heavy-metal zinc, which may be associated with intracellular granules [33 ]. Because PKC is a zinc metalloenzyme, the possible effects of zinc preferentially binding to the PKC and affecting the PKC location were examined. However, inclusion of the heavy-metal chelator, TPEN, during cell preparation did not alter the location of PKC {alpha} (Fig. 5B) .

The effect of treatment of eosinophils with PMA at a concentration that ensures maximal superoxide production in these cells (1 µM) [7 ] was also assessed. However, the treatment did not cause the relocation of PKC {alpha}, although activation is inferred by the rapid down-regulation of PKC in the particulate fraction (Fig. 6A ). The unusual PKC location and behavior are confirmed through a subsequent similar experiment in which the effect of PMA treatment on PKC {alpha} location and level in equine neutrophils was determined. PKC is located in the cytosol in untreated neutrophils, and a rapid translocation to the membrane is observed after 5 min of PMA treatment (Fig. 6B) . Partial down-regulation of PKC is seen by 20 min in these cells.



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  		Figure 6. Effect of PMA on the location and amount of PKC {alpha} in equine eosinophils and neutrophils. A) Untreated eosinophils or cells maintained in buffer with PMA for 5 and 20 min were separated into detergent-soluble (lanes 1) and -insoluble (lanes 2) fractions before analysis by Western blotting. B) Untreated neutrophils or cells maintained in buffer with PMA for 5 and 20 min were separated into cytosol (lanes 1), membrane (lanes 2), and particulate (lanes 3) fractions before analysis by Western blotting. The position of PKC {alpha} is indicated. The insoluble fraction in A corresponds to the particulate fraction in B, and the detergent-soluble fraction in A has been divided into cytosol and membrane fractions in B.

 
PKC activity
Use of the conventional PKC substrate histone IIIS identified cofactor-dependent PKC activity in the detergent-insoluble, but not the soluble, eosinophil fraction (Fig. 7A ). However, using the {delta} pseudosubstrate-site peptide, activity was found to be present in the detergent-soluble and -insoluble cell fractions. The PKC activity present in the detergent-soluble cell fraction was cofactor-independent, whereas phosphatidylserine and PMA stimulated the activity in the detergent-insoluble cell fraction (Fig. 7B) . Phosphorylation of substrates in the absence of cofactor and PMA indicates the presence of a phosphatidylserine (PS)- and PMA-independent kinase activity, which may be PKC that has been partially deregulated during purification and is a normal feature of this type of assay, or a different protein kinase altogether. Alternatively, the PKC may be partially stimulated by copurified phospholipids. We have not differentiated between these alternatives.



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  		Figure 7. PKC activity in equine eosinophils. A) Activity measured using the substrate histone IIIS. Detergent-soluble and detergent-insoluble cellular fractions of equine eosinophils were incubated with histone IIIS and [32P-{gamma}] ATP with or without the PKC cofactors PS and PMA, as indicated. Phosphotransfer in control samples (extraction buffer) in the absence and presence of cofactors is also indicated. Values are expressed as mean of triplicate determinations. B) Activity measured using the substrate {delta} pseudosubstrate-site peptide. The same cellular fractions of equine eosinophils and the control buffer were incubated with {delta} pseudosubstrate-site peptide and [32P-{gamma}] ATP with or without the PKC cofactors PS and PMA, as indicated. Values are expressed as mean of triplicate determinations. The results in A and B are those of a single experiment, and similar data were generated in a second study.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Studies of the effects of PKC inhibitors in human and guinea pig eosinophils have implicated PKC in the regulation of a range of eosinophil functional responses to different mediators. There is also limited evidence indicating that individual isotypes might be involved in eosinophil activation [17 , 34 ]. PMA, which is known to activate PKC in many different cells [21 , 22 , 25 , 35 36 37 38 ], increases superoxide production by equine eosinophils and can stimulate the adherence of these cells to protein-coated plastic [7 , 10 ]. The present study has shown that PKC inhibitors such as staurosporine and bisindolymaleimide I inhibit PMA-induced superoxide production by equine eosinophils. These findings, taken together with data suggesting that PKC is involved in eosinophil function in other species [14 15 16 17 18 19 20 , 39 ] and in pore fusion in equine eosinophils [23 ], led us to characterize the PKC isotypes in equine eosinophils and to measure the activity of the PKC that was present.

Because none of the PKC isotype-selective antibodies that were available for use had been raised using horse proteins, species cross-reactivity was first established using equine brain tissue, a rich source of PKC [26 ]. Selectivity of binding was also confirmed using the epitope peptides, because the antibodies recognize PKC isotypes by specific sequences near the carboxyl termini [24 ]. A similar profile of PKC isotypes (conventional PKC {alpha}, ß, and {gamma}, novel PKC {delta} and {varepsilon}, and atypical PKC {iota} and {zeta}) was found to be present in equine and rat brain, indicating that the antibodies recognized equine PKC. The absence of any reactive bands on the Western blots following incubation with antibody plus epitope peptides also demonstrated selective and specific binding of the antibodies in those cases where peptide was available.

Equine eosinophils contained PKC isotypes {alpha}, ß, {delta}, {varepsilon}, {iota}, and {zeta}. Interestingly, the majority of the PKC in equine eosinophils was recovered in the detergent-insoluble particulate fraction. This conflicts with data obtained using human eosinophils and neutrophils. PKC {alpha}, ßI, ßII, {delta}, {varepsilon}, {iota}, and {zeta} have been reported to be present in human eosinophils, but the majority of the PKC was present in the cytosol, the remainder being located in the membrane [34 ]. Human neutrophils have been shown to express PKC {alpha}, ßI, ßII, {delta}, and {zeta}, and again the isotypes were present in the cytosol [35 , 40 41 42 ]. In agreement with data obtained using human granulocytes and in contrast to our findings using equine eosinophils, we show that the PKC in equine neutrophils was primarily located in the cytosol.

One possible explanation for the unusual location of equine eosinophil PKC could be the presence of zinc [33 ]. A correlation has been found between the concentration of zinc and the presence of PKC in the cytoskeleton of HL60 cells [43 ]. If zinc were present in greater amounts in equine eosinophils than in eosinophils from other species, this might induce PKC to localize with the cytoskeleton, leading to its recovery in the particulate fraction on purification. However, inclusion of TPEN, a heavy-metal chelator, at a concentration that effectively binds zinc [30 ], during the purification process, did not affect the location of equine eosinophil PKC {alpha} within the cell. Another possible explanation could be that degradation of PKC was initiated during cell purification. Degradation follows a requisite dephosphorylation of the enzyme, which is followed by translocation to the cytoskeleton [32 ]. However, as with TPEN, no change in the intracellular location of PKC {alpha} was observed when the phosphatase inhibitor, okadaic acid, was included during cell separation at a concentration that has been shown to inhibit PKC protein movement through the exocytic pathway of intact Chinese hamster ovary (CHO) cells [29 ]. Thus, the location of equine eosinophil PKC may be similar to that found in bovine brain in which the PKC is associated with the nucleus in a "membrane-inserted" form [44 ] and could be important for relaying responses to and from the nucleus [45 , 46 ]. In agreement with this suggestion, further fractionation of equine eosinophils localized PKC mainly to the nuclear particulate fraction, with a small amount of the enzyme associated with the soluble nuclear fraction and the cytoskeleton. Further studies are clearly required to establish the significance of the localization of PKC in equine eosinophils.

The PKC in equine eosinophils could be activated, suggesting that it was not in the process of degradation. The down-regulation response to PMA treatment infers activation, because PKC down-regulation is thought to require PKC activation as a prerequisite [47 ]. The normal location and behavior of PKC {alpha} in equine neutrophils upon PMA treatment further confirm the abnormal findings in the eosinophil. The activity assays also demonstrate that PKC, even in the particulate fraction of equine eosinophils, can be activated. Cofactor-dependent PKC activity was detected in the particulate fraction of the cell when histone IIIS and the {delta} pseudosubstrate-site peptide were used as substrates, indicating the presence of a PKC. The presence of kinase activity in the detergent-soluble cell fraction detected using the {delta} pseudosubstrate-site peptide could be a result of a novel PKC because this substrate, but not histone IIIS, is phosphorylated by novel PKCs. Because there are no readily available substrates to assay for these PKC isotypes specifically at present, this cannot be confirmed.

Because PKC activation by PMA in equine eosinophils leads to the respiratory burst, and PKC has been identified in these cells, it will be of interest in future studies to determine the effect of PKC inhibition on such functional responses as activation, adherence, and migration in response to different mediators. Evidence of a regulatory role for individual PKC isotypes in human eosinophils has been obtained, which suggests that PKC {delta} and {varepsilon} are involved in adhesion-dependent NADPH oxidase activation [17 ]. More direct evidence of mediator-induced activation of PKC isotypes has been obtained using human neutrophils, formyl-methionyl-leucyl-phenylalanine (fMLP), having been shown to cause translocation of PKC {delta} from the cytosol to the membrane [35 ]. Opsonized zymosan has also been reported to cause the translocation of PKC ßII, {delta}, and {zeta} to the membrane and {delta} and {zeta} to the granule fraction in these cells [48 ]. Mediator-induced activation, as well as an antisense approach, which has been used to show that selective depletion of PKC ß inhibits fMLP-induced free-radical generation in HL60 cells differentiated to a neutrophil-like phenotype [49 ], will be applied to equine eosinophils to provide further insight into the role of each of the identified PKC isotypes in regulating the function of these cells.


   ACKNOWLEDGEMENTS
 
E. C. G. is a BBSRC training scholar.

Received December 30, 1999; revised May 13, 2000; accepted May 14, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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