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Eur Respir J 2001; 18:130-138
Copyright ©ERS Journals Ltd 2001

Expression of members of the phospholipase A2 family of enzymes in human nasal mucosa

J. Lindbom, A.G. Ljungman, M. Lindahl and C. Tagesson

Dept of Health and Environment, Division of Occupational and Environmental Medicine, Faculty of Health Sciences, University of Linköping, S-581 85, Linköping, Sweden

CORRESPONDENCE: A.G. Ljungman, Dept of Health and Environment, Division of Occupational and Environmental Medicine, Faculty of Health Sciences, University of Linköping, S-581 85, Linköping, Sweden. Fax: 46 13145831

Keywords: Inflammation, nasal mucosa, phospholipase A2, polymerase chain reaction

Received: June 19, 2000
Accepted March 3, 2001

This work was supported by the Swedish Medical Research Council (K99-27X-05983-19A), the Swedish Council for Work Life Research (96-0615) and the Vårdal Foundation, Sweden (96/48 A96 027).


   Abstract
TOP
 Abstract
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Phospholipase A2 (PLA2) is a family of enzymes thought to play a key role in inflammation by releasing arachidonic acid for the synthesis of eicosanoids and lysophospholipid for the synthesis of platelet-activating factor. However, the precise contribution of different PLA2 types to the formation of inflammatory lipid mediators in the upper airways is not known and the expression of different PLA2 genes in the human nasal mucosa has not been examined.

This study therefore investigated the occurrence of messenger ribonucleic acids (mRNAs) for different PLA2 forms (IB, IIA, IID, IIE, III, IVA, IVB, IVC, V, VI, VII, X, acid calcium-independent (aiPLA2), and calcium-independent membrane bound PLA2, (iPLA2-2)) in the nasal mucosa of five healthy human subjects.

Using reversed transcription-polymerase chain reaction (RT-PCR) techniques it was found that all these PLA2 types except PLA2 V were expressed in all subjects, whereas PLA2 V was detected in only one individual on one single occasion. The relative abundance of the different PLA2 transcripts were aiPLA2>X{approx}IVA>IIA{approx}IIE{approx}IVB{approx}VI>IB{approx}IID{approx}III{approx}IVC{approx}VII{approx}iPLA2-2. To further quantify the mRNA-expression of PLA2 X, IVA and IIA, the samples were reanalysed with a quantitative PCR-technique utilizing competitive deoxyribonucleic acid (DNA) mimics as references. The amounts of PLA2 X, IVA and IIA mRNA were then estimated to 0.9±0.2, 1.1±0.7, and 0.0025±0.0021 amol (mean±se), respectively, confirming the relative abundance of these PLA2 transcripts and indicating that the recently described PLA2 X form is relatively strongly expressed.

These findings demonstrate that a large number of PLA2 types are expressed in the normal human nasal mucosa. Moreover, this investigation demonstrates, for the first time, the presence of the newly discovered phospholipase A2 forms IID, IIE, III, IVB, IVC, X and calcium-independent membrane bound phospholipase A2 in the human nasal mucosa and raises the possibility that one or several of these may be involved in inflammatory reactions in the nose.

Phospholipase A2 (PLA2) is the first enzyme in the synthesis of two types of lipid inflammatory mediators with potent effects in the respiratory tract: the eicosanoids and platelet-activating factor (PAF) 1. Accordingly, PLA2 hydrolyzes the acyl-ester bond at the sn-2 position of glycerophospholipids and releases arachidonic acid (AA), which is then metabolized to eicosanoids, and 1-O-alkyl-lysophosphatidylcholine (lysoPAF), which is the precursor of PAF. At present, a number of different PLA2s (IB, IIA, IID, IIE, III, IVA, IVB, IVC, V, VI, VII, X and the cytosolic calcium-independent membrane bound PLA2 (iPLA2-2)) have been identified in human tissues 2–6. These PLA2s belong to different types of enzymes 12: the secretory type (sPLA2), the cytosolic calcium-dependent type (cPLA2), and iPLA2 8. However, the precise contribution of these different PLA2 types to the formation of inflammatory lipid mediators in the airways is not known, and it may well be that yet other proteins with PLA2 properties are present. It is important, therefore, to study in more detail the diversity of PLA2s in the airways and how the different PLA2s may be activated during inflammation.

Despite the general interest in different PLA2 types and how they participate in the release of lysoPAF and AA, the role of different PLA2s in the human nasal mucosa has received rather little attention. Previous investigations have demonstrated increased PLA2 activity in nasal lavage fluid (NLF) from allergic patients after allergen provocation 10, 11; although the increased PLA2 activity was not unequivocally attributed to any specific PLA2 type, both investigations highlighted an sPLA2 with characteristics similar to PLA2 IIA. High concentrations of a PLA2 IIA have also been determined in NLF from healthy subjects after methacholine provocation 12; however, the PLA2 levels in NLF were low after obstruction of the flow of tear fluid into the nasal cavity, indicating that the enzyme originated from tear fluid. In addition to PLA2 IIA, Touqui et al. 11 found PAF acetylhydrolase-like activity in NLF, indicating the presence of type VII sPLA2. Despite these original and important findings, it is still unclear how the different PLA2s participate in the formation and metabolism of lipid mediators in the nasal mucosa. Moreover, a large number of novel PLA2 types have recently been isolated and cloned 3–6, 13–17, many of which may turn out to be operating in the nasal mucosa or in NLF. These considerations prompted the present effort to examine the occurrence of messenger ribonucleic acids (mRNAs) for different PLA2 forms in the nasal mucosa of healthy human subjects.


   Materials and methods
TOP
 Abstract
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects
Five subjects volunteered for the investigation. They were all healthy males, 25–45 yrs of age, nonsmokers and not taking any drugs. All five subjects were used to obtain nasal brush samples while three of them were used to obtain NLF samples. Brush samples were collected at different occasions from each subject with no less than a week between sampling occasions. All individuals were examined clinically at each sampling occasion, visually checking (by anterior rhinoscopy) that the nasal cavity appeared normal. Thus, the nasal mucosa did not show any signs of inflammation or irritation before nasal lavage or brushing.

Nasal mucosal samples
Cells from the nasal mucosa were obtained either by nasal lavage or by a brush technique as previously described 18. To obtain NLF, 15 mL of saline were inserted by means of a syringe connected to a commercial Foley catheter (Bard Ltd, Crawley, West Sussex, UK) with the cuff adapted in the vestibulum nasi. The saline was recovered from the nasal cavity after 10 min and the cell content was collected by centrifugation. To obtain nasal brush samples, small plastic-coated, steel-wire brushes with nylon bristles were employed. The brush was placed between the septum and inferior turbinate and rotated while being removed. Cells obtained by brushing were immediately collected by twisting the brush in 1 mL of phosphate buffered saline at pH 7.3 followed by centrifugation. The total number of cells in the nasal brush and NLF samples were 2.7x105±2.1x105 and 2.3x104±1.5x104, respectively. The viability of the cells exceeded 97%, in both preparations, as determined by Tryptan blue exclusion. Differential countings stained with Wright stain (Accustain, Sigma-Aldrich, St Louis, MO, USA) showed that the mucosal samples obtained by either method consisted mainly of epithelial cells. An average of 5.6±1.8% of the total number of cells were leukocytes and among these only occasional eosinophils were detected.

Detection of messenger ribonucleic acid by reverse transcriptase-polymerase chain reaction
The cell pellets were immediately lysed and total cellular ribonucleic acid (RNA) isolated by the method of acid guanidium thiocyanate/phenol/chloroform extraction as previously described 19. RNA was reverse-transcribed into complementary DNA (cDNA) with Moloney Murine Leukaemia Virus (MMLV) Reverse Transcriptase (RT) (SuperScriptTM II, Life Technologies Ltd, Paisley, UK) according to the manufacturers instructions. Briefly, total RNA was mixed with a buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.6 mM of each deoxyribonucleotide triphosphate (dNTP), and 0.5 µg oligonucleotide (deoxythumidylate)18 (oligo (dT)18), and heated for 5 min at 65°C. After the incubation, the samples were chilled on ice and RT SuperScriptTM II enzyme (200 units), RNAsine (20 units) and dithiothreitol (DTT) (100 mM) was added in a final volume of 20 µL. The samples were incubated for 60 min at 40°C followed by 5 min at 95°C. The cDNAs were stored at –20°C until used in PCR amplifications.

For polymerase chain reaction (PCR), cDNA from all subjects were pooled and 1 µL of the pooled cDNA was used per PLA2 form amplified. All PCR reactions contained 2 µL PCR buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.6), 2 mM MgCl2, 0.2 mM dNTP mix, 0.1 mM oligonucleotide primers, and 0.5 units Taq DNA polymerase in a total volume of 20 µL. For the amplification of PLA2 IIA and IV, Platinum Taq DNA polymerase (Life Technologies) was used. All primers (table 1Go) used were constructed from published sequences and then synthesized by Life Technologies. The PCR reactions were carried out in a PTC-100 (MJ Research Inc., Watertown, MA, USA) programmable thermal controller with an initial 2 min denaturation at 94°C followed by the cycled program 1 min at 94°C, 1 min at 55–65°C (varying between primer sets) and 1 min at 72°C. All PCR reactions were carried out for 40 cycles (figs. 1–5GoGoGoGoGo) except the competitive analyses of PLA2 IVA and X which were carried out for 28 cycles (figs. 6–7GoGo). A final extension of 7 min at 72°C ended the reaction. Ten microlitres from each PCR reaction was separated on a 1.5% agarose gel and ethidium bromide-stained. The stained gels were digitized into gray-scale images with a DC120 Zoom Digital Camera (Kodak Digital Science, Rochester, NY, USA) and analysed with the Kodak Digital Science lD Image Analysis Software. The PCR products obtained corresponded to the expected product sizes as listed in table 1Go. The PCR products were confirmed by Southern blot using oligonucleotide probes complementary to sequences within the different PLA2 PCR products (the oligonucleotide probes used are listed in table 1Go) or by restriction enzyme digestion using PVU II (isolated from Proteus vularis) for the IID, IVB and aiPLA2 forms, Hha I (isolated from Haemophillus haemidyticus) for the IIE form, Sty I (isolated from Salmonella Typhi) for the III form and Hind III (isolated from Haemophillus influence Rd) for the iPLA2-2 form (Life Technologies).


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  		Table 1— Description of primers used in polymerase chain reactions and digonucleotides used in southern blots.

 


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  		Fig. 1.— Gene expression of different phospholipase A2 (PLA2) types in nasal mucosal cells obtained through lavage. Total ribonucleic acid isolated from the cells was converted to complementary deoxyribonucleic acid through reverse transcription, and analysed by polymerase chain reaction for the IB, IIA, IID, IIE, III, IVA, IVB, IVC, V, VI, VII, X, and calcium-independent membrane bound PLA2 (aiPLA2; ai) and calcium-independent membrane bound PLA2 (iPLA2-2; i-2) types. S: 100 base pair size marker.

 


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  		Fig. 2.— Gene expression of different phospholipase A2 (PLA2) types in nasal mucosal cells obtained by brushing. Total ribonucleic acid (RNA) isolated from the cells was converted to complementary deoxyribonucleic acid through reverse transcription, and analysed by polymerase chain reaction (PCR) for the IB, IIA, IVA, IVC, V, VI, VII, and X types (left panels a, c). The 1,000 base pair band visible in the Southern blot for the V form (d) is probably due to genomic contamination. The expected PCR-product size for each PLA2 type is indicated. S: 100 base pair size marker. The right panels (b, d) show Southern blot confirmations of the PLA2 PCR fragments utilizing {gamma}-32P-labelled oligonucleotide probes.

 


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  		Fig. 3.— Gene expression of phospholipase A2 (PLA2) IID, IIE, IVB and acid calcium-independent membrane bound PLA2 in nasal mucosal cells obtained by brushing. Total ribonucleic acid isolated from the cells was analysed by reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products were treated with PVU II (PLA2 IID, IVB and aiPLA2), Hha I (PLA2 IIE) or Sty I (PLA2 III). This way, the 328 base pair PLA2 IID product was cut into 259 and 69 bp fragments, the 324 bp PLA2 IIE product into 207 and 117 bp fragments, the 266 bp PLA2 III product into 191 and 65 bp fragments, the 272 bp PLA2 IVB product into 174 and 98 bp fragments, the 451 bp aiPLA2 product into 250 and 201 bp fragments, and the 303 calcium-independent membrane bound PLA2 (iPLA2-2) product into 211 and 92 bp fragments, as predicted by the restriction enzyme map. –: uncut fragment; +: cut fragment; S: 100 base pair size marker.

 


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  		Fig. 4.— Expression of different phospholipase A2 (PLA2) types in nasal mucosal cells obtained by brushing. A 10-fold dilution series of pooled complimentary deoxyribonucleic acid was analysed by polymerase chain reaction.

 


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  		Fig. 5.— a) Competitive polymerase chain reaction (PCR) analysis of the phospholipase A2 (PLA2) IIA(276 base pairs (bp)) messenger ribonucleic acid (mRNA) level in nasal mucosal cells obtained by brushing. Total RNA was used as template for complementary deoxyribonucleic acid (cDNA) synthesis and a portion of the cDNA was amplified in the presence of different amounts of a PLA2 IIA mimic (327 bp). Lanes 2–9 contain decreasing concentrations of PLA2 IIA mimic, and lane 1 contains a 100 bp size marker. b) The quantitative analysis of the competitive PCR shown in a): y=–0.26x–0.83; R2=0.97.

 


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  		Fig. 6.— Competitive polymerase chain reaction (PCR) analysis of the A2 (PLA2) IVA (341 base pairs (bp)) messenger ribonucleic acid (mRNA) level in nasal mucosal cells obtained by brushing. Total RNA was used as template for complementary deoxyribonucleic acid (cDNA) synthesis and a portion of the cDNA was amplified in the presence of different amounts of a PLA2 IVA mimic (292 bp). Lanes 2–7 contain decreasing concentrations of PLA2 IVA mimic, and lane 1 contains a 50 bp size marker with the 250 bp band enhanced. b) The quantitative analysis of the competitive PCR shown in (a): y=–0.2x–0.15; R2=0.87.

 


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  		Fig. 7.— a) Competitive polymerase chain reaction (PCR) analysis of the phospholipase A2 (PLA2) X (295 base pairs (bp)) messenger ribonucleic acid (mRNA) level in nasal mucosal cells obtained by brushing. Total RNA was used as template for complementary deoxyribonucleic acid (cDNA) synthesis and a portion of the cDNA was amplified in the presence of different amounts of a PLA2 X mimic (243 bp). Lanes 1–7 contain decreasing concentrations of PLA2 X mimic and lane 8 contains a 50 bp size marker with the 250 bp band enhanced. b) The quantitative analysis of the competitive PCR shown in a): y=–0.32x–0.44; R2=0.98.

 
To allow a comparison of the relative abundance of the PLA2 forms, the amplification efficiency (E) was determined for each primer set. Aliquots of cDNA were subjected to varying numbers of amplification cycles and the PCR product was treated as described earlier. The cDNA amounts (expressed as net intensities) from respective number of cycles were then plotted as the log (amount PLA2 form) versus number of cycles. After linear regression, the E value was deduced from the slope of the semi-log plot. The E values were: PLA2 IB (E=0.68), IIA (E=0.77), IID (E=1.19), IIE (E=0.68), III (E=0.56), IVA (E=0.84), IVB (E=0.53), IVC (E=1.13), V (E=not determined), VI (E=0.90), VII (E=0.67), X (E=0.64), iPLA2-2 (E=0.88), and aiPLA2 (E=0.53).

Quantitative polymerase chain reaction
In order to further quantify the amounts of mRNA, competitive DNA fragments (PCR mimics) were constructed for the PLA2 forms IIA, IVA and X. The PCR mimics were generated by two successive PCR amplifications of heterologous DNA (pGEM® plasmide control DNA, Promega Corp., Madison, WI, USA). For the first PCR, composite primers were used. One contained the upstream target primer (PLA2 IIA, IVA or X; see table 1Go) linked to a primer that anneals to the heterologous DNA. The other contains the downstream PLA2 target primer and the oligonucleotide sequence for the PLA2 target (table 1Go) linked to the opposite strand of the heterologous DNA. The second PCR is performed with the PLA2-specific primer. The PLA2 mimics thus generated contain the target sequences for the PLA2-specific primers and the target sequence for the internal oligonucleotide probe, allowing confirmation of the specific PLA2 product as well as the mimic product by Southern blot. The internal control fragments (mimics) and cDNA fragments from the PLA2 forms were simultaneously amplified to ensure a parallel accumulation over the whole cycle range and a similar E-value for the accumulation for the two fragments. Due to large E-value differences between the cDNA PLA2 IVA and its mimic, a new antisense primer was constructed giving a 341 bp PCR product (fig. 6Go). The E-values were: mimic PLA2 IIA and cDNA PLA2 IIA 0.81 and 0.78, mimic PLA2 IVA and cDNA PLA2 IVA 0.71 and 0.72, and mimic PLA2 X and cDNA PLA2 X 0.59 and 0.53, respectively. The amounts of PCR product generated in the coamplification of cDNA from a sample with different amounts of mimic was used to determine the level of specific PLA2 mRNA present in the sample. The amounts of the two products separated on an agarose gel (Life Technologies) ethidium bromide stained, digitized into gray-scale images and expressed as net intensity were plotted as the log (PLA2-form/mimic) versus log (mimic concentration). After linear regression and expression in the format y=kx+l, the amount of PLA2 in the sample was calculated from y=0 (figs. 5–7GoGoGo). The intra-assay and interassay variation of the determinations were tested by amplifying a dilution series of the PLA2 mimic with 0.1 amol, respectively, 1 amol of PLA2 cDNA added to each dilution. The PCR reactions were performed in triplicate at three different occasions. The mean±sd values for the detected amounts of PLA2 cDNA in the nine determinations were for the 0.1 amol samples: PLA2 IIA 0.07±0.02 amol, PLA2 IVA 0.29±0.04 amol, PLA2 X 0.10±0.02 amol, and for the 1 amol samples: PLA2 IIA 1.08±0.36 amol, PLA2 IVA 2.99±0.92 amol, and PLA2 X 1.20±0.25 amol.


   Results
TOP
 Abstract
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Expression of phospholipase A2 in human nasal mucosal cells
The expression of different PLA2 forms in NLF and nasal brush samples are illustrated in figures 1 and 2GoGo. It thus appeared that the IB, IIA, IID, IIE, III, IVA, IVB, IVC, V, VI, VII, X, iPLA2-2 and aiPLA2 forms were all expressed in the mucosal cell samples, regardless of sampling technique. The different PCR products were confirmed using Southern blot and {gamma}-32P-oligonucleotides (fig. 2Go), or by use of the restriction enzymes PVU II, Hha I or Sty I (fig. 3Go). Repeated experiments showed essentially the same pattern of PLA2 expression, except that PLA2 V mRNA was not detected; this particular PLA2 form was thus found only in pooled samples from the first sampling round. Analysis of mRNA from the five different subjects revealed that the PLA2 V expression originated from one single individual. Repeated samples were taken from this individual and analysed; however, no PLA2 V mRNA was found on these later occasions. By contrast, all the other PLA2 forms were found to be expressed in all subjects, including the individual that expressed the PLA2 V form on one single occasion.

To investigate the relative abundance of the different PLA2 mRNA forms, a ten-fold dilution series of pooled cDNA was analysed (fig. 4Go). This revealed that aiPLA2 was detected in the 1:104 dilution, followed by PLA2 IVA and X in the 1:103 dilution, IIA, IIE, IVB, and VI in the 1:102 dilution, and IB, IID, III, IVC, VII, and iPLA2-2 in the 1:10 dilution. The large amounts of aiPLA2 mRNA detected were not due to a more efficient amplification reaction; in fact, the PCR for this PLA2 type had the lowest E-value of all.

Quantitative messenger ribonucleic acid analysis of phospholipase A2 IIA, IVA and X
In order to quantitatively assess the expression of PLA2 IIA, IVA and X mRNA, competitive PCR analyses were performed using mimics for these forms. The application of these mimics is illustrated in figures 5–7GoGoGo, which show the measurements of PLA2 IIA, IVA and X mRNA in a nasal mucosal sample. After linear regression analysis, the amounts of mRNA in this specific sample were calculated to be 6.8x10–3, 0.18 and 0.05 amol, respectively.

The mimics were then used to determine the amounts of PLA2 IIA, IVA and X mRNA in nasal mucosal samples from the five subjects. After normalization to their reduced glyceraldehyde-phosphate-dehydrogenase (GAPDH) level the amounts of PLA2 IIA, IVA and X mRNA were estimated to 0.0025±0.0021, 1.1±0.7, and 0.9±0.2 amol (mean±se), respectively (table 2Go). The relative abundance of these PLA2 transcripts (as demonstrated in fig. 4Go) was thus confirmed.


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  		Table 2— Competitive polymerase chain reaction (PCR) analysis of the phospholipase A2 (PLA2) IIA, IVA and X messenger ribonucleic acid (RNA) levels in nasal mucosal cells from five healthy subjects.

 

   Discussion
TOP
 Abstract
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
The present investigation showed that PLA2 IB, IIA, IID, IIE, III, IVA, IVB, IVC, VI, VII, X, iPLA2-2 and aiPLA2 mRNA were expressed in the human nasal mucosa in all subjects investigated. This study thus not only confirmed the presence of PLA2 IIA 12 and VII 11 but also demonstrated the presence of a large number of other members of the PLA2 superfamily, including the newly described PLA2 IID 14, IIE 4, III 6, IVB 16, IVC 17, X 20, and iPLA2-2 3, 5. It should be emphasized, however, that the presence of PLA2 gene transcripts does not necessarily mean that the mRNA is translated into proteins or that these proteins, if present, are activated PLA2 enzymes. Further studies must be undertaken in order to reveal which PLA2 enzyme proteins are operating in the nasal mucosa.

Previous studies have identified some of the different PLA2 types in other parts of the human respiratory tract, such as PLA2 IB 21, IIA 12, IID 14, IIE 4, VII 22, 23, and X 20. PLA2 III was cloned from the foetal lung, but Northern blot did not reveal any transcript in the lung 6. Cytosolic PLA2 IVA has been identified in human alveolar macrophages and respiratory cell lines 24, 25, and the recently discovered paralogues, PLA2 IVB and IVC, in human lung tissue 16, 17. By contrast, cytosolic PLA2 VI (iPLA2) has, to the present authors' knowledge, not been demonstrated in human airways until now. PLA2 VI mRNA corresponds to an 85 kDa murine PLA2 that has been identified in P388D1 cells 26. Although this enzyme shares no homology with the calcium-dependent PLA2 IVA and possesses no clear preference for AA-containing phospholipids, it is thought to play a role in the remodelling of membrane phospholipid and has been shown to be of importance in regulating the AA incorporation into membranes 2. It is possible, therefore, that this enzyme plays an important part in the overall AA metabolism.

As illustrated in figure 4Go, there were striking differences in the relative abundance of the different PLA2 types. The strongest expression was shown by aiPLA2, which has been proposed to play a major part in the recycling of lung surfactant, as it has preference for dipalmitoyl phosphatidylcholine 9, 27. However, it is questionable whether this enzyme is a true PLA2 and if it has PLA2 properties at physiological pH. The aiPLA2 also has glutathione peroxidase activity, and has instead been proposed as an antioxidative protein 28. Interestingly, PLA2 X was also relatively strongly expressed (fig. 4Go), indicating that this PLA2 type is abundantly present in the nasal mucosa. The results obtained with the mimic technique confirmed the order of appearance of PLA2 X, IVA and IIA shown by the dilution series, with PLA2 X and IVA being about 400-fold more strongly expressed than PLA2 IIA (table 2Go). There is therefore reason to believe that PLA2 X is strongly expressed in the human nasal mucosa.

PLA2 X is a low molecular weight (13.6 kDa), calcium-dependent, secretory PLA2 that contains 16 cysteine residues 8, 29. Because it binds to a PLA2 lung membrane receptor 30, it is thought to play a role in cell signaling. PLA2 X has been detected in human adult lung 20 and type II alveolar epithelial cells 31 but not in the human nasal mucosa until now. Interestingly, NLF from individuals exposed to cold air or methacholine has high PLA2 activity. A previous study using a fluoroimmunoassay has suggested that this activity is due to PLA2 IIA derived from tear fluid 12. In the same study, however, immunostaining of nasal biopsies showed only few PLA2 IIA-positive cells, which is in accordance with the present findings that the mRNA expression of PLA2 IIA is weak. Altogether, these findings suggest that PLA2 X, rather than PLA2 IIA, should be considered as the major sPLA2 in the nasal tract of healthy individuals. The function of PLA2 X is not known. Notably, it has been demonstrated that PLA2 X releases significantly larger amounts of AA than do other sPLA2 forms (IB, IIA and V) when added exogenously to adherent cells 8, 29, 31, suggesting that PLA2 X may play an important role in AA liberation.

As to which PLA2 type(s) are involved in airway inflammation and disease, much attention has been given to the secretory (14 kDa)-type (sPLA2). The PLA2 IIA has been connected to inflammatory diseases such as Crohn's disease and endotoxic shock 32. This form is inducible by endotoxin in rats 14, 33 and in guinea pigs 34 and has also been proposed to take part in the defence against bacterial infection 35. The recently discovered sPLA2 II forms, IID and IIE, might also take part in the bacterial defence, as both are able to hydrolyze phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the major components of bacterial phospholipids.

Aside from the sPLA2 group, cPLA2 has also been considered important for inflammatory signaling 36. The present results showed that the expression of PLA2 IVA mRNA was greater than that of PLA2 IIA in the nasal mucosa of healthy subjects. This cPLA2 is known to be of importance for AA liberation with the most convincing results obtained through experiments with PLA2 IVA deficient mice. Such studies have revealed that the IVA form is of importance in the allergic response and for macrophage production of inflammatory mediators 36, 37. This suggests a role for PLA2 IVA in airway inflammation. In addition to PLA2 IVA, it was also found that mRNA of the two novel IVB and IVC forms in the nasal mucosa. Biochemical characterization by Song et al. 38 has shown that IVA has a preference for the sn-2 position and the IVB for the sn-1 position, while IVC enzymes can cleave efficiently well at both positions. The IVA and IVB are both calcium-dependent enzymes while IVC is not. This indicates different biological roles or regulations of these cPLA2 forms.

During recent years another sPLA2 (type V) has been implicated in inflammatory signalling and it has been suggested that some of the results concerning PLA2 IIA should be reconsidered in the light of the presence of PLA2 V 7. In the present study the V form was only detected in one out of five healthy individuals. Repeated efforts to demonstrate PLA2 V in mucosal samples from this one individual were unsuccessful, indicating that the expression was occasional and that this form was induced by some stimuli at that specific time point. Further studies are needed to investigate if this PLA2 form perhaps is more highly expressed in patients with airway inflammation.

Taken together, the issue of PLA2 activity and regulation is growing more complex. New PLA2 forms with unknown functions, cross-talking between different PLA2s 39, splice variants of certain PLA2s 15, and a large number of proteins that may affect PLA2 activity 13, 40, 41 all add up to a picture of great complexity. It is important, however, to gain a deeper knowledge about the role and regulation of each PLA2 and so to be able to develop new and more specific anti-PLA2 drugs. In light of the present study the nasal mucosa offers a location for further studies, in which most, if not all the PLA2 forms are expressed.

In conclusion, it has been found that a large number of phospholipase A2 types are expressed in the normal human nasal mucosa. Moreover, the investigation demonstrates for the first time, the presence of the newly discovered phospholipase A2 forms IID, IIE, III, IVB, IVC, X and the cytosolic calcium-independent membranebound phospholipase A2 (iPLA2-2). The precise function of each of these phospholipase A2 forms is not clear. The nasal mucosa is constantly exposed to environmental factors such as particles, chemicals, allergens and micro-organisms and the presence of messenger ribonucleic acid of several phospholipase A2 forms may therefore reflect a need for a fast response and a first line of defence towards all these stimuli. It is likely, however, that one or several of these PLA2 enzymes are important to the arachidonic acid and/1-O-alkyl-lysophosphatidylcholine release in the nasal mucosa. Future studies aiming at identifying the molecular mechanisms underlying inflammatory reactions or diseases of the nasal mucosa will therefore have to take these findings into consideration.


   Acknowledgements
TOP
 Abstract
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors wish to thank K. Irander for performing the clinical examination.


   References
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 Abstract
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Barnes PJ, Chung KF, Page CP. Inflammatory mediators of asthma: an update. Pharmacol Rev 1998;50:515–596.[Abstract/Free Full Text]
  2. Balsinde J, Balboa M, Insel P, Dennis E. Regulation and inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol 1999;39:175–189.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  3. Mancuso DJ, Jenkins CM, Gross RW. The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A2. JBC 2000;275:9937–9945.[Abstract/Free Full Text]
  4. Suzuki N, Ishizaki J, Yokota Y, et al. Structures, enzymatic properties and expression of novel human and mouse secretory phospholipase A2. JBC 2000;275:5785–5793.[Abstract/Free Full Text]
  5. Tanaka H, Takeya R, Sumimoto H. A novel intracellular membrane-bound calcium-independent phospholipase A2. Biochem Biophys Res Commun 2000;272:320–326.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  6. Valentin E, Ghomashchi F, Gelb M, Lazdunski M, Lambeau G. Novel human secreted phospholipase A2 with homology to the group III bee venom enzyme. JBC 2000;275:7492–7496.[Abstract/Free Full Text]
  7. Dennis EA. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 1997;22:1–2.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  8. Murakami M, Kambe T, Shimbara S, et al. Different functional aspects of the group II subfamily (types IIA and V) and type X secretory phospholipase A2s in regulating arachidonic acid release and prostaglandin generation. JBC 1999;274:31435–31444.[Abstract/Free Full Text]
  9. Fisher AB, Dodia C. Role of acidic Ca2+-independent phospholipase A2 in synthesis of lung dipalmitoyl phosphatidylcholine. Am J Physiol 1997;272:L238–L243.
  10. Stadel JM, Hoyle K, Naclerio RM, Roshak A, Chilton FH. Characterization of phospholipase A2 from human nasal lavage. Am J Respir Cell Mol Biol 1994;11:108–113.[Abstract]
  11. Touqui L, Herpin-Richard N, Gene R-M, et al. Excretion of platelet activating factor-acetylhydrolase and phospholipase A2 into nasal fluids after allergenic challenge: possible role in the regulation of platelet activating factor release. J Allergy Clin Immunol 1994;94:109–119.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  12. Aho HJ, Grénman R, Sipilä J, Peuraavuori H, Hartikainen J, Nevalainen TJ. Group II phospholipase A2 in nasal fluid, mucosa and paranasal sinuses. Acta Otolaryngol 1997;117:860–863.[Medline] [Order article via Infotrieve]
  13. Clark MA, Conway TM, Shorr RGL, Crooke ST. Identification and isolation of a mammalian protein which is antigenically and functionally related to the phospholipid A2 stimulatory peptide melittin. JBC 1987;262:4402–4406.[Abstract/Free Full Text]
  14. Ishizaki J, Suzuki N, Higashino K, et al. Cloning and characterization of novel mouse and human secretory phospholipase A2s. JBC 1999;274:24973–24979.[Abstract/Free Full Text]
  15. Larsson PKA, Claesson HE, Kennedy BP. Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity. JBC 1998;273:207–214.[Abstract/Free Full Text]
  16. Pickard TR, Strifler BA, Kramer RM, Sharp JD. Molecular cloning of two new human paralogs of 85-kDa cytosolic phospholipase A2. JBC 1999;274:8823–8831.[Abstract/Free Full Text]
  17. Underwood KW, Song C, Kriz RW, Chang XJ, Knopf JL, Lin L-L. A novel calcium-independent phospholipase A2, cPLA2-{gamma}, that is prenylated and contains homology to cPLA2. JBC 1998;273:21926–21932.[Abstract/Free Full Text]
  18. Irander K, Stahlbom B, Welinder H, Akesson B. Metachromatic cells and eosinophils in the nasal mucosa and N,N-dimethylbenzylamine exposure. Acta Otolaryngol 1997;117:433–436.[Medline] [Order article via Infotrieve]
  19. Ljungman AG, Tagesson C, Lindahl M. Endotoxin stimulates the expression of group II PLA2 in rat lung in vivo and in isolated perfused lungs. Am J Physiol 1996;270:L752–L760.
  20. Cupillard L, Koumanov K, Mattéi MG, Lazdunski M, Lambeau G. Cloning, chromosomal mapping, and expression of a novel human secretory phospholipase A2. JBC 1997;272:15745–15752.[Abstract/Free Full Text]
  21. Seilhamer JJ, Randall TL, Yamanaka M, Johnson LK. Pancreatic phospholipase A2: Isolation of the human gene and cDNAs from porcine pancreas and human lung. DNA 1986;6:;5: 519–527.
  22. Chilton FH, Averill FJ, Hubbard WC, Foneth AN, Triggiani M, Liu MC. Antigen-induced generation of lyso-phospholipids in human airways. J Exp Med 1996;183:2235–2245.[Abstract/Free Full Text]
  23. Triggiani M, De Marino V, Sofia M, et al. Characterization of platelet-activating factor acetylhydrolase in human bronchoalveolar lavage. Am J Respir Crit Care Med 1997;156:94–100.[Abstract/Free Full Text]
  24. Shamsuddin M, Chen E, Anderson J, Smith L. Regulation of leukotriene and platelet-activating factor synthesis in human alveolar macrophages. J Lab Clin Med 1997;130:615–626.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  25. Wu T, Ikezono T, Angus WC, Shelhamer JH. Tumor necrosis factor-{alpha} induces the 85-kDa cytosolic phospholipase A2 gene expression in human bronchial epithelial cells. Biochim Biophys Acta 1996;1310:175–184.[Medline] [Order article via Infotrieve]
  26. Ackermann E, Kempner E, Dennis E. Ca(2+)-independent cytosolic phospholipase A2 from macrophage-like P338D1 cells. Isolation and characterization. JBC 1994;269:9227–9233.[Abstract/Free Full Text]
  27. Kim T, Sundaresh C, Feinstein S, et al. Identification of a human cDNA clone for lysosomal type Ca2+-independent PLA2 and properties of the expressed protein. JBC 1997;272:2542–2550.[Abstract/Free Full Text]
  28. Phelan S, Johanson K, Beier D, Paigen B. Characterization of the murine gene encoding Aop2 (antioxidant protein 2) and identification of two highly related genes. Genomics 1998;54:132–139.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  29. Bezzine S, Koduri R, Valentin E, et al. Exogenously added human group X secreted phospholipase A2 but not the group IB, IIA and V enzymes efficiently release arachidonic acid from adherent mammalian cells. JBC 2000;275:3179–3191.[Abstract/Free Full Text]
  30. Yokota Y, Higashino K, Nakano K, Arita N, Hanasaki K. Identification of group X phospholipase A2 as a natural ligand for mouse phospholipase A2 receptor. FEBS 2000;478:187–191.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  31. Hanasaki K, Ono T, Saiga A, et al. Purified group X secretory phospholipase A2 induced prominent release of arachidonic acid from human myeloid leukemia cells. JBC 1999;274:34203–34211.[Abstract/Free Full Text]
  32. Kaiser E. Phospholipase A2: Its usefulness in laboratory diagnostics. Crit Rev Clin Lab Sci 1999;36:65–163.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  33. Ljungman A, Lindahl M, Tagesson C. Asbestos fibers and manmade mineral fibers: induction and release of tumor necrosis factor-alpha from rat alveolar macrophages. Occup Environ Med 1994;51:777–783.[Abstract]
  34. Arbibe L, Vial D, Rosinski-Chupin I, et al. Endotoxin induces expression of type II Phospholipase A2 in macrophages during acute lung injury in guinea pigs. J Immunol 1997;159:391–400.[Abstract]
  35. Weinrauch Y, Abad C, Liang N, Lowry S, Weiss J. Mobilization of potent plasma bactericidal activity during systemic bacterial challenge. J Clin Invest 1998;120:633–638.
  36. Bonventere JV, Huang Z, Taheri MR, O'Leary E, Moskowitz MA, Sapirstein A. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 1997;390:622–625.[CrossRef][Medline] [Order article via Infotrieve]
  37. Uozomi N, Kume K, Nagase T, et al. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 1997;390:618–622.[CrossRef][Medline] [Order article via Infotrieve]
  38. Song C, Chang XJ, Bean K, Proia M, Knopf J, Kriz R. Molecular characterization of cytosolic phospholipase A2-ß. JBC 1999;274:17063–17067.[Abstract/Free Full Text]
  39. Balsinde J, Balboa MA, Dennis EA. Functional coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2. Proc Natl Acad Sci USA 1998;95:7951–7956.[Abstract/Free Full Text]
  40. Russo-Marie F. Annexin V and phospholipid metabolism. Clin Chem Lab Med 1999;37:287–291.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  41. Jorens PG, Sibille Y, Goulding NJ, et al. Potential role of Clara cell protein, an endogenous phospholipase A2 inhibitor, in acute lung injury. Eur Respir J 1995;8:1647–1653.[Abstract]



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B. P. Hurley and B. A. McCormick
Multiple Roles of Phospholipase A2 during Lung Infection and Inflammation
Infect. Immun., June 1, 2008; 76(6): 2259 - 2272.
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