MAST CELL MARKERS

Although many mast cell mediators or products serve as useful markers of mast cell activation in vitro, it has been notoriously difficult conclusively to establish mast cell activation in human studies. For example, it is difficult to catch the short lived increase in plasma levels of histamine and its metabolites following allergen induced bronchoconstriction. Furthermore, circulating basophils may contribute significantly to plasma histamine⁹ and plasma values may be increased by non-specific challenges such as an ordinary exercise test. Measurements of urinary metabolites of histamine may sometimes be helpful to provide information regarding systemically released histamine over time¹⁰ but, due to extensive metabolism, only a small percentage of circulating histamine levels appear in the urine and the ambiguity with regard to the cellular source remains.

Tryptases, which are proteases secreted by degranulating human mast cells, have been reported to make up about 25% of total mast cell protein.¹¹ This would seem to make tryptase an ideal marker of mast cell activation. Although tryptase measurements are very useful in experimental work with cells and tissues, this marker has not been particularly helpful in mechanistic studies addressing mast cell activation in humans. This may relate to limitations in the currently available methodology for measuring plasma or serum tryptase. Nevertheless, so far, the main uses of tryptase measurements are to provide evidence for the diagnosis of systemic mastocytosis or necropsy evidence of systemic anaphylaxis.¹²

Prostaglandins (PG) are ubiquitously biosynthesised and would therefore seem to be unlikely candidates as specific markers for any particular cell. However, in this issue of Thorax, Bochenek et al¹³ confirm and extend the accumulated evidence that measurement of PGD₂ or its metabolites represents a sensitive and reliable strategy for assessment of mast cell activation in vivo. Specifically, they convincingly show, for the first time, increased levels of the primary PGD₂ metabolite 9α11β-PGF₂ in plasma during the early phase of allergen induced airway obstruction. This is achieved by applying gas chromatography-negative ion chemical ionisation-mass spectrometry (GC-NICI-MS) to samples collected at frequent intervals before and during allergen bronchoprovocation of subjects with atopic asthma. The methodology is very appropriate as GC-NICI-MS is the most specific measurement of this particular family of compounds, where the presence of numerous structurally related metabolites always complicates immunoassay measurements. Bochenek et al also deserve credit for their development of a protocol that improves the sensitivity of the GC-NICI-MS measurements.

BIOSYNTHESIS OF PGD₂ IN MAST CELLS

The release of PGD₂ from isolated human mast cells was reported more than two decades ago,¹⁴ shortly followed by the demonstration of its release into human airways after local endotracheal instillation of allergen.¹⁵ However, the mechanistic significance of these reports was not generally appreciated. In humans, mast cells are an almost exclusive cellular source of PGD₂.¹⁶ Although there is evidence of some PGD₂ formation by platelets, macrophages and certain T lymphocytes,¹³ the reported amounts are 100–1000 times lower than those produced during IgE dependent activation of mast cells. More importantly, whereas the basophil and the mast cell both release histamine and leukotriene (LT) C₄, it is only the mast cell that produces significant quantities of PGD₂.¹⁶ There is, in fact, recent evidence to show that increased expression of the haematopoetic PGD₂ synthase may be the functional response that is most specifically upregulated in activated mast cells.¹⁷

MEASUREMENT OF PGD₂

The currently renewed interest in applications of PGD₂ measurement would not have been possible without the comprehensive work of Roberts and colleagues at Vanderbilt who performed painstaking GC/MS identifications of PGD₂ metabolites in blood and urine after injections of radiolabelled PGD₂.^18,19 More than 25 metabolites were identified but intact PGD₂ was not found in the urine. The most abundant PGD₂ metabolite identified was 9,11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioc acid, commonly referred to as PGD-M. The earliest appearing urinary metabolite was 9α,11β-PGF₂, which was subsequently shown to be stereospecifically transformed from PGD₂ by the NADPH dependent enzyme 11-ketoreductase²⁰ in lung and liver. Interestingly, 9α,11β-PGF₂ retains biological activity. It has, for example, been found to contract bronchial smooth muscle²¹ and has vascular effects including contraction of coronary arteries.²² Metabolism of 9α,11β-PGF₂ by the 15-hydroxy prostaglandin dehydrogenase, followed by β- and ω- oxidations, leads to PGD-M.

The Vanderbilt group thus used GC/MS measurements of PGD-M as a marker of systemic PGD₂ production in different disease states. Markedly raised levels of PGD-M were discovered in systemic mastocytosis²³ as well as during anaphylaxis. The GC/MS approach is, however, laborious and technologically demanding, which generally renders it less applicable to studies of populations and large numbers of samples. The more recent validation of an immunoassay method for the measurement of 9α,11β-PGF₂ in urine^24,25 has therefore created new opportunities for using this PGD₂ metabolite as a mast cell marker. Using this immunoassay methodology, increased excretion of metabolites of PGD₂ into the urine has been observed after allergen induced bronchoconstriction^10,24 and mast cell involvement in other indirect challenges has also been confirmed.^24,26,27

As discussed by Bochenek et al,¹³ apart from weak indirect or anecdotal evidence, there has not previously been any investigation of 9α,11β-PGF₂ levels in plasma during allergen induced bronchoconstriction, which undoubtedly must be the gold standard for mast cell activation. Interestingly, the current demonstration of increased PGD₂ release during allergen induced bronchoconstriction puts further weight behind a previous publication from the group in Krakow where increased plasma levels of 9α,11β-PGF₂ were found following aspirin induced bronchoconstriction.²⁸ This adds to several other lines of evidence²⁴ suggesting that the intolerance to aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) involves mast cell activation.

Bochenek et al confirmed the original observations by Liston et al¹⁸ that 9α,11β-PGF₂ was the first PGD₂ metabolite to appear in urine, although they did not find the increase of this metabolite in the urine to be as great as that reported by O’Sullivan et al.¹⁰ These seemingly different findings are most probably explained by the demonstration²⁵ that the immunoassay measures not only 9α,11β-PGF₂ but also at least two other metabolites that appear somewhat later. In other words, for detection of the sum of the initially excreted PGD₂ metabolites in urine, the chemically less specific immunoassay will paradoxically have greater practical sensitivity as it measures several related PGD₂ metabolites. However, as pointed out by Bochenek et al, for studies of the kinetics of 9α,11β-PGF₂ metabolism, the chemically more specific method is obviously preferable.

PERSPECTIVES

The method chosen to monitor mast cell activation by measurement of PGD₂ metabolites will obviously depend on the questions asked and the resources available. Irrespective of the analytical method selected, measurements of 9α,11β-PGF₂ in plasma, urine, or other body fluids currently provide the most sensitive method for detection of mast cell activation in vivo. This was clearly shown in the paper by Bochenek et al, where there was no change in plasma tryptase despite the fivefold increase in plasma 9α,11β-PGF₂. Similarly, in previous work by O’Sullivan et al,^10,26 there was consistently a much smaller or non-significant increase in urinary methyl histamine in contrast to consistent and prominent increases in urinary 9α11β-PGF₂ metabolites. Thus, for investigations into the role of the mast cell in different pulmonary diseases, measurements of PGD₂ metabolites in body fluids offer many new opportunities.

Finally, PGD₂ is not only a marker of mast cell activation but also—together with its immediate metabolite 9α,11β-PGF₂—it is a potent mediator of bronchoconstriction, vasomotor tone, and cell recruitment.²⁹ We hypothesise that PGD₂ mediates the component of allergen induced bronchoconstriction that remains resistant to antihistamines and antileukotrienes.³⁰ Experimental data are available to support such a role,^29,31 and a role for PGD₂ in rhinitic responses in humans has also been implicated.³² The recent awareness that there are at least three different receptors (TP, DP, and CRTH2) mediating the effects of PGD₂ in the airways²⁹ suggests that we may soon get improved opportunities to define more precisely the pulmonary role of this mast cell derived mediator.