RATIONALE: Assays for tryptase have proved useful in the laboratory diagnosis of anaphylaxis, but in a substantial number of cases increased circulating concentrations of this mast cell product are not detected. The measurement of levels of mast cell carboxypeptidase, a protease co-released with tryptase, could provide information useful in diagnosis. METHODS: A sandwich-based ELISA procedure with specific monoclonal antibodies has been developed and validated for the measurement of carboxypeptidase. This assay has allowed the release of carboxypeptidase to be detected in supernatants from experimentally activated mast cells. It has been adapted and applied to determine concentrations in serum and plasma from cases of suspected anaphylaxis (n=181), systemic mastocytosis (30), and from control groups consisting of healthy blood donors (209) or subjects with bronchial asthma (15). RESULTS: Carboxypeptidase levels in serum or plasma collected within 8h of the onset of an allergic reaction were significantly greater than in those of either of the control groups (p<0.0001). In some cases the concentrations were more than 100-fold greater. Levels in the mastocytosis group were also greater than in the control groups (p<0.001). There was no significant difference between the two control groups. In 83% of anaphylaxis cases with an elevated tryptase concentration there were carboxypeptidase levels greater than the normal range, though concentrations of these mast cell proteases were not correlated with each other. Out of 110 cases of suspected anaphylaxis that were tryptase negative, there was an elevated concentration of carboxypeptidase in 77 (70%). CONCLUSIONS: Mast cell carboxypeptidase should be a valuable serum marker for anaphylaxis.

Matrix metalloproteinases (MMPs) are involved in a number of physiological and pathologic processes including the inflammation found in 1131). We have shown that MMP-3 is upregulated in Crohn's disease and in ulcerative colitis. This study shows a potential role for MMP-12 in these idiopathic diseases.

RATIONALE: Mast cell subpopulations have frequently been categorized according to the expression of the serine proteases tryptase and chymase. Carboxypeptidase is established as a major constituent of mast cell secretory granules, but the distribution of this protease in human mast cell populations has been little examined. METHODS: A series of monoclonal antibodies were prepared against mast cell carboxypeptidase purified from human tissues, and employed in immunohistochemistry to examine the distribution of this protease in two conditions associated with mast cell infiltration of the affected tissues: bronchial asthma and osteoarthritis. RESULTS: The new antibodies reacted in ELISA and in immunoblotting with carboxypeptidase purified from human skin or from recombinant expression systems. There was selective binding to mast cells in immunohistochemistry with a range of tissues. Immunohistochemistry with double labeling or the application of camera lucida techniques with sequentially cut thin sections indicated that cells staining for carboxypeptidase were generally stained also for tryptase and for chymase. Cells staining for carboxypeptidase represented a small proportion of those staining for tryptase in the bronchial mucosa and there was little difference in numbers between asthmatic and non-asthmatic subjects. In non-diseased synovial tissues, some 20% of mast cells expressed both of these proteases, but in tissues from cases of osteoarthritis the proportion was much higher (80%; p<0.009). Relative numbers of carboxypeptidase and chymase-staining cells were similar in all tissues examined. CONCLUSIONS: Mast cell carboxypeptidase appears to be restricted to the chymase-containing subset of human tissues in health and disease.

Rationale TGF-β exists in an inactive complex form bound to tissue proteoglycans, such as decorin, and requires release to exert its biological effects. Plasmin, a product of plasminogen activation, has been implicated in activating latent TGF-β and will be generated in association with tissue inflammation. We investigated the effects of plasmin on cytokine and TGF-β1 release and gene expression in primary fibroblast cultures from asthmatics and healthy controls. Methods Fibroblasts were grown from endobronchial biopsies of volunteers and used at passages 3-5. They were serum deprived for 24 hours, then challenged with plasmin for a further 24 hours. Cell supernatants were assayed for growth factors and cytokines by ELISA. Fibroblast mRNA was reverse transcribed and subjected to real-time PCR using Taqman probes. Results Plasmin induced a concentration-dependent (P Conclusions Plasmin promoted the liberation of TGF-β1, a cytokine associated with remodeling, but did not induce pro-inflammatory cytokines. The plasmin-induced release of immunomodulatory TGF-β1 may serve to regulate the inflammatory response and its effects on structural cells will promote tissue remodeling.

Tryptase, the major secretory product of human mast cells, is emerging as a new target for therapeutic intervention in allergic airways disease. We have investigated the ability of tryptase and inhibitors of tryptase to modulate histamine release from human lung mast cells and have examined the potential contribution of proteinase-activated receptor 2 (PAR2). The tryptase inhibitor APC366 [N-(1-hydroxy-2-naphthoyl)-l-arginyl-l-prolinamide hydrochloride] was highly effective at inhibiting histamine release stimulated by anti-IgE antibody or calcium ionophore from enzymatically dispersed human lung cells. A concentration of APC366 as low as 10 μM was able to inhibit anti-IgE-dependent histamine release by some 50%. Addition of leupeptin or the tryptic substrate N-benzoyl-d,l-arginine-p-nitroanilide also inhibited IgE-dependent histamine release. Purified tryptase in the presence of heparin stimulated a small but significant release of histamine from lung cells, suggesting that tryptase may provide an amplification signal from activated cells that may be susceptible to proteinase inhibitors. Trypsin was also able to induce histamine release apparently by a catalytic mechanism. Moreover, pretreatment of cells with metabolic inhibitors or with pertussis toxin reduced responses, indicating a noncytoxic pertussis toxin-sensitive G proteinmediated signaling process. Addition to cells of the PAR2 agonists SLIGKV-NH2 or tc-LIGRLO-NH2 or appropriate control peptides were without effect on histamine release, and PAR2 was not detected by immunohistochemistry in tissue mast cells. The potent actions of tryptase inhibitors as mast cell-stabilizing agents could be of value in the treatment of allergic inflammation of the respiratory tract, possibly by targeting the non-PAR2-mediated actions of tryptase.

RATIONALE: Basogranulin is a constituent of basophil secretory granules that has been identified using the basophil-specific monoclonal antibody BBI.We have investigated the kinetics of basogranulin secretion from experimentally activated basophils, and compared them with the release of histamine and tryptase.METHODS: Basophils were purified by immunomagnetic selection from peripheral blood and stimulated to degranulate with a range of concentrations of anti-IgE antibody, calcium ionophore A23187, fMLP, and C5a.Basogranulin release was quantified by a dot blotting procedure.Histamine concentrations were measured by a glass microfiber-based method, and tryptase by determining cleavage of a chromogenic substrate.RESULTS: Basogranulin, histamine and tryptase were released in response to anti-lgE antibody with bell-shaped concentration response curves.This pattern was seen also for histamine and tryptase.With A23187, fMLP and C5a, these mediators were secreted in a purely concentration-dependent manner.Wortmannin suppressed anti-IgE antibody induced basophil activation.Basogranulin release in response to anti-lgE was observed after 30 s stimulation and maximal release (28%) was achieved by 15 min.In all studies, the kinetics of basogranulin secretion appeared similar to those for histamine with levels closely correlated (p<0.0001).Basophils from some donors contained little tryptase, but levels of this protease also correlated with those of basogranulin (p<0.0001).CONCLUSIONS: Basogranulin is secreted together with histamine and tryptase, and with similar kinetics of release, in response to various stimuli of basophil degranulation.The measurement of this unique basophil marker should be valuable in distinguishing basophil activation from that of mast cell activation.

Inhibitors of mast cell tryptase and chymase can be effective as mast cell stabilising compounds. Lactoferrin has been reported to inhibit tryptase activity, but its actions on other serine proteases of mast cells and its potential to alter mast cell function are not known. We have examined the ability of lactoferrin to inhibit mast cell tryptase, chymase and cathepsin G, and investigated its potential to modulate the activation of human mast cells. Enzymatically dispersed cells from human skin, lung and tonsil were challenged with anti-IgE or calcium ionophore A23187, following incubation with recombinant human lactoferrin, and histamine release determined. IgE-dependent histamine release from skin mast cells was inhibited by up to 50% following incubation with lactoferrin (50 or 500 nM). Tonsil mast cells were also stabilised by lactoferrin, but not those from lung. Calcium ionophore A23187-induced histamine release was not altered by lactoferrin. A double-labelling immunocytochemical procedure revealed the presence of lactoferrin in 4–6% of mast cells, and this proportion increased to 40% following incubation with lactoferrin. Lactoferrin did not inhibit cleavage of synthetic substrates by tryptase and chymase directly, though it was able to diminish the ability of heparin to stabilise tryptase. Cathepsin G activity was inhibited by lactoferrin. The ability of lactoferrin to inhibit IgE-dependent activation of human mast cells and modulate protease activity suggests that the release of this neutrophil product may have a role in the downregulation of allergic inflammation.

The basophil was first described in the peripheral blood by Ehrlich more than one hundred years ago [1]. For much of the time that has elapsed since, this cell type has languished in the shadow of its tissue-resident counterpart: the mast cell. Basophils have been characterized as a rich source of histamine, eicosanoids, cytokines and other mediators, but they are the rarest of leucocytes, normally accounting for less than one percent of those leucocytes found in blood [2, 3]. There have long been reports that the numbers of these cells may be increased in the peripheral blood in allergic disease, or that they may migrate into the affected tissues. However, much of the evidence for basophil accumulation in tissues has been circumstantial, and research in this field has come to be neglected. Only recently have appropriate tools become available that allow the reliable detection of basophils in human tissue. Histochemical staining with basic dyes, the mainstay of basophil detection procedures since the time of Ehrlich, are generally unsuitable for tissues routinely processed with formaldehyde-based fixatives, and do not allow basophils to be distinguished reliably from tissue mast cells. The dye is taken up by proteoglycans in the secretory granules of both of these cell types, and it is a major challenge to discern the subtle differences in the relative size and numbers of granules, or in the shape of the nucleus. At the ultrastructural level, the larger size of basophil granules, and the segmental nucleus of this cell type is more apparent [4], but electron microscope studies covering small areas of tissue are not suitable for generating quantitative data. In the absence of a unique marker for basophils, some workers have employed a double labelling procedure to identify cells strongly expressing the high affinity IgE receptor, but which fail to express the mast cell marker tryptase [5]. The value of such approaches as a means for identifying basophils has been undermined by the recognition not only that various other cell types may express FcεRI [6, 7] but also that cells of the peripheral blood (presumably basophils) may sometimes express quite high levels of tryptase [8]. Advances in the understanding of the biology of human mast cells occurred in the late 1980s and in the 1990s with the development of monoclonal antibodies specific for tryptase [9, 10], other proteases of the mast cell granule [9, 11, 12], and for the membrane marker c-kit[13]. A basophil-specific antibody was produced by Bodger et al. as long ago as 1987. This antibody, termed Bsp-1, binds in flow cytometry to a 45-kDa cell surface protein expressed by basophils [14] and a proportion of KU812 cells (a line with basophilic properties [15]), but is generally unsuitable for the immunohistochemical identification of basophils in tissues. Another antibody, named 97A6, is directed against a cell surface antigen that is reported to be expressed more abundantly following basophil activation [16]. However, this antigen is expressed to some extent also by mast cells and its potential as a marker for basophils remains to be clarified. Two monoclonal antibodies that recognize granule proteins specific for basophils are now showing great promise as a means for the identification of this cell type. These antibodies, termed 2D7 [17] and BB1 [18], bind to proteins of unknown function that are secreted upon degranulation. Antibody 2D7 binds to a protein of 72–76 kDa [17] that is produced at an early stage in basophil development from bone marrow progenitors [19]. BB1 antibody recognizes a highly basic protein, termed basogranulin, that appears to exist as a large macromolecular complex of approximately 5000 kDa or more, and is secreted along with histamine upon cell stimulation [18, 20]. The development of specific immunoassays for basogranulin and the 2D7 antigen, and elucidation of their mediator roles, could one day provide important clues as to what may be the unique contribution of basophils in allergic disease. In the meantime, BB1 and 2D7 are providing a means for the reliable identification of basophils in tissues for the first time and are allowing relative numbers of this cell type to be determined in allergic conditions. In the absence of allergen exposure, relatively few basophils appear to be present in the tissues of allergic subjects. Immunostaining with BB1 antibody has revealed that basophils are rare in nasal biopsies taken from subjects with seasonal allergic rhinitis out of season [21]. In the lower airways, basophils detected by the same technique are slightly more numerous in bronchial biopsies from asthmatic subjects than in those from control subjects, but they are much less common than either mast cells or eosinophils [22]. In unstimulated skin of atopic subjects, basophils are rarely, if ever, observed [23]. Following experimental allergen challenge, basophil numbers may increase dramatically in all of these tissues. Intranasal administration of allergen can induce the rapid influx of basophils into the nasal mucosa of rhinitic subjects [21]. This response can be evident as early as one hour after allergen challenge, and significant basophilia can last for at least a week afterwards. There have been close parallels in asthmatic subjects, with increases in the basophil numbers in bronchial biopsies observed in response to inhalation challenge [22, 24], and a substantial proportion of these cells have been noted to possess IL-4 [24]. In the skin also, allergen-provoked reactions have been found to be associated with basophil infiltration into the inflamed area after challenge [22, 25]. Diffuse staining of extracellular material reacting with both 2D7 and BB1 has been taken as indicative of basophil degranulation at these sites. The kinetics of basophil recruitment following allergen challenge is quite different from that of eosinophils or neutrophils, suggesting different processes involved in cell recruitment. Thus, for example, Ying and colleagues have reported that basophil numbers are maximal at 24 h when eosinophil numbers have declined from their 6-h maximum [23]. In that study there was a clear association between the degree of eosinophil infiltration at 6 h and eotaxin expression in the tissues, and at 24 h with eotaxin-2 and monocyte chemotactic protein 4 (MCP-4) expression; whereas expression of none of these CC chemokines, nor of RANTES or MCP-3, was related to the numbers of basophils at the 24-h peak. In the model of Fahy et al. [26], in which human skin has been grafted onto severe combined immunodeficiency (SCID) mice reconstituted with human mononuclear cells, injection of eotaxin or RANTES, but not monocyte-derived chemokine (MDC) or IP-10, has been found to stimulate the influx of BB1-reactive cells [26]. Processes of basophil recruitment are likely to be complex, and could differ between clinical conditions and tissue compartments. Increased numbers of basophils in the affected tissue following experimental allergen challenge have now been reported in several studies. The effect of chronic natural allergen exposure on numbers of tissue basophils, or the effect of treatment, has not hitherto been reported. In a recent issue of Clinical and Experimental Allergy, Wilson et al. describe the results of a study in which basophils were enumerated in nasal tissue collected from subjects with seasonal allergic rhinitis outside the pollen season and from the same individuals two years later at the height of the pollen season [27]. Over this period, subjects received a course of allergen-specific immunotherapy or placebo. In untreated subjects, basophils as detected immunohistochemically with 2D7 antibody were found to be more numerous in both the submucosa and epithelium in tissues collected during the pollen season. In keeping with previous studies, seasonal increases in numbers of eosinophils and mast cells, but not neutrophils, were noted in biopsy tissues. Basophil infiltration into the epithelium during the pollen season appeared to be reduced by allergen-specific immunotherapy, an observation made also for eosinophils. This study thus provides further evidence for a role for basophils in the pathogenesis of seasonal allergic rhinitis, and the authors suggest that immunotherapy may work, at least in part, by reducing the recruitment of basophils to the nasal tissues. This is an exciting time for basophil research. We now have confirmation that appreciable numbers of these cells can be recruited into the tissues following experimental or natural chronic exposure to allergen, and that immunotherapy may be associated with reduced numbers. There is still much to learn about the underlying processes of basophil accumulation and how recruitment and activation may be modulated by immunotherapy and other treatments. The availability of the new basophil-specific antibodies is opening up new avenues for investigation, and should allow a better evaluation of the true roles of these neglected cells in allergic disease.

Although asthma has been viewed mainly as an eosinophilic disease, and chronic obstructive pulmonary disease (COPD) as a neutrophilic disease, recent studies have shown increased neutrophil counts in severe asthma and sputum eosinophilia in some COPD patients. In an attempt to further characterise these two syndromes according to pathology, the current authors have conducted a study of induced sputum in 15 subjects with COPD, 17 asthmatics, and 17 nonatopic healthy individuals. Sputum was analysed for cytology and levels of eosinophil cationic protein (ECP), albumin, tryptase and soluble intercellular adhesion molecule‐1. The COPD subjects differed from the asthmatics as they had higher sputum neutrophil and lower columnar epithelial cell counts, but there were no differences in any soluble marker studied. When compared to control subjects, both the asthmatic and COPD subjects had raised eosinophil counts and ECP levels. In a subset of COPD subjects with sputum eosinophilia (>3% of total cells), significantly increased levels of tryptase were detected. In conclusion, although chronic obstructive pulmonary disease is a more neutrophilic disease than asthma, the two diseases are difficult to distinguish on the basis of sputum levels of the soluble markers traditionally associated with asthma. However, a subset of patients with chronic obstructive pulmonary disease with airway eosinophilia and mast-cell activation might represent a distinct pathological phenotype.

Clinical & Experimental Allergy ReviewsVolume 1, Issue 2 p. 68-72 Roles of the mast cell and basophil in asthma A F Walls, A F Walls Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this authorS He, S He Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this authorM G Buckley, M G Buckley Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this authorA R McEuen, A R McEuen Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this author A F Walls, A F Walls Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this authorS He, S He Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this authorM G Buckley, M G Buckley Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this authorA R McEuen, A R McEuen Immunopharmacology Group, University of Southampton School of Medicine, Southampton, UKSearch for more papers by this author First published: 07 July 2008 https://doi.org/10.1046/j.1472-9725.2001.00009.xCitations: 24 Dr. Andrew F.Walls, Immunopharmacology Group, Mailpoint 837 Level F, South Block, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume1, Issue2July 2001Pages 68-72 RelatedInformation

Recently, certain chemokines and chemokine receptors have been preferentially associated with the selective recruitment in vitro of type 1 T cells, such as IP-10 and its receptor CXCR3, or type 2 T cells such as monocyte-derived chemokine (MDC) and eotaxin and their receptors CCR4 and CCR3. Very few models have provided confirmation of these findings in vivo. Taking advantage of the humanized SCID mouse model grafted with autologous human skin, the ability of the chemokines IP-10, MDC, eotaxin, and RANTES to stimulate cell recruitment was investigated. Intradermal IP-10 injection resulted in an influx of CD4+ T lymphocytes but also surprisingly in the recruitment of dendritic cells. MDC recruited mainly CD8+ T lymphocytes, and had little effect on eosinophils. As predicted, eotaxin was a potent inducer of eosinophil and basophil migration, also recruiting CD4+ T cells. RANTES, a ubiquitous chemokine associated with both type 1 and type 2 profiles, was able to recruit all cell types. CXCR3-positive cells were preferentially recruited by IP-10, whereas CCR3- and CCR4-positive cells were predominantly found after injection of eotaxin and MDC. Thus, in a human environment in vivo, some chemokines have the ability to recruit cells expressing chemokine receptors preferentially expressed on type 1 or type 2 cells. Further investigations revealed that MDC and eotaxin induced the recruitment of type 2, but not type 1, cytokine-producing cells. RANTES, on the other hand, induced the migration of both type 1 and type 2 cytokine-secreting cells, whereas IP-10 did not induce the recruitment of either subtype. These studies provide detailed information on the properties of MDC, eotaxin, IP-10, and RANTES as chemotactic molecules in skin in vivo. The use of the humanized SCID mouse model grafted with human skin is validated as a useful model for the evaluation of chemokine function in the inflammatory reaction, and suggests that therapeutic targeting of certain chemokines might be of interest in diseases associated preferentially with a type 1 or type 2 profile.

BACKGROUNDBB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils.OBJECTIVEWe sought to further characterize the basophilspecific antigen identified by BB1.METHODSIntracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing.RESULTSIn flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 x 10(6) d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6.CONCLUSIONWith its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein.

Background Sudden Infant Death Syndrome, (SIDS) or cot death, remains the most common category of post-perinatal death in the UK. By definition, the cause of death is unknown, but a long-standing theory is that some of these deaths could be the result of anaphylaxis.Objective To investigate the potential contribution of anaphylactic mechanisms to deaths in infancy by determining relative levels of alpha- and beta -tryptases and both total and allergen-specific IgE in sera from groups of infants whose deaths were attributed to SIDS or to other causes.Methods Serum samples were collected at the time of post-mortem examination from infants whose death was classed as SIDS (n=40) and from a comparison group in which cause of death had been established (n=32). Serum tryptase concentrations were measured with a radioimmunoassay with monoclonal antibody G5 which detects primarily beta -tryptase or an ELISA with antibody AA5 which has equal sensitivity for alpha- and beta -tryptases. Levels of total IgE and IgE specific for casein, beta -lactoglobulin, house dust mite and moulds were determined.Results Analysis of the results of the two assays for tryptase indicated that levels of the beta -like tryptase (the form secreted on anaphylactic degranulation) were significantly higher in serum from infants with SIDS compared with those whose death was explained. There was no evidence for an increase in serum levels of alpha -tryptase (the variant secreted constitutively from mast cells). Total levels of serum IgE did not differ between the two groups and, reflecting the low circulating IgE concentrations in infancy, an elevation in IgE specific for the panel of allergens was not detected.Conclusions In a proportion of SIDS victims there may be increased serum levels of beta -like tryptase, a marker for anaphylaxis. The failure to detect an increase in alpha -tryptase would suggest that mast cell hyperplasia is not a feature of cot death. The nature of the inciting agents remains unclear, but anaphylaxis deserves serious consideration as a possible cause of sudden death in infancy.