Mast cell isolation

M, Marone G Human heart mast cells. Isolation, purification, ultrastructure and immunologic characterization. J Immunol 1995 154 2855. 28... 

Rees, P.H., Hillier, K. and Church, M.K. (1988). The secretory characteristics of mast cells isolated from the human large intestinal mucosa and muscle. Immunology 65, 437-442. 

The importance of mast cell-sensory nerve interactions in human airways remains to be established. There are, to our knowledge, no data regarding the ability of mast cell products to stimulate C-fibers in human airways. Substance P does cause the degranulation of human skin mast cells (Lawrence et /., 1987), and injection of substance P into human skin causes itching and a wheal-and-flare response (Wallengren and Hakanson, 1987). However, mast cells isolated from human limg tissue obtained at surgical resection do not release H when exposed to substance P (Lawrence et cd., 1987). 

Also of interest is the observation that protein Fv is a potent inducer of mediator release from mast cells isolated from human heart tissue [58,63]. This represents the first evidence that a human protein induced by viral infections can act as an endogenous immunoglobulin superantigen causing the release of vasoactive and immunoregulatory mediators from cardiac mast cells. 

L stimulates in vitro the release of preformed and de novo synthesized vasoactive and proinflammatory mediators (histamine, tryptase and cysteinyl leukotriene) by human mast cells isolated from heart tissue [45], As shown in figure 1, protein L binds ex vivo to B cells expressing human k L-chain-positive Igs. 

The histamine content of isolated HHMC (=3 pg/cell) was comparable to lung parenchymal and skin mast cells. The mean tryptase content of HHMC (=24 pg/10 cells)... 

Neurotensin (NT) is a tridecapeptide (Table 4.2) first isolated from brain and gut by Carraway and Leeman [75] and reported by them to induce a rapid and transient hypotension, a cutaneous vasodilatation, and a cyanosis of the extremities in the anaesthetized rat. This report, along with others [76-78] indicating that the NT-induced hypotension and increased vascular permeability could be blocked by histamine receptor antagonists such as mepy-ramine [77] or by pretreatment with compound 48/80 [76], suggested that endogenous histamine (perhaps released from tissue mast cells) was involved in producing some of the biological effects of NT [78]. 

NT was initially reported to stimulate the non-cytotoxic release of histamine from isolated rat peritoneal mast cells by workers in our laboratory [40, 79]. This observation has now been confirmed and extended by several other workers [80-85]. When added to isolated rat serosal mast cells, NT initiates the secretion of histamine which is dependent upon calcium and energy [79]. Secretion begins at about 10 nanomolar NT and reaches an initial plateau of some 20% histamine release at 10 piM NT [79] (Figure 4.2) while higher levels... 

Interestingly, pretreatment of isolated rat peritoneal mast cells with NT desensitizes the cells to the subsequent stimulation by compound 48/80 (Figure 4.3). However, the desensitized cells are still responsive to stimulation by the calcium ionophore, A23187, in the presence of Ca (Figure 4.3). A similar observation has been made when compound 48/80 and the peptides,... 

Some workers have reported a failure of NT to elicit significant histamine release (< 5% at 10 /xM NT) from isolated peritoneal mast cells obtained from Wistar strain rats [82]. In these studies, when NT was added to mast cells from the pleural cavity [82] or when the C-terminal octapeptide (NT6 13) or the C-terminal hexapeptide (NT8 13) was added to peritoneal mast cells, a significant (>20% histamine release) secretory response occurred [82]. In our laboratory a significant difference in the responsiveness to NT of peritoneal and pleural mast cells from Sprague-Dawley rats has also been found, pleural cells eliciting a higher percentage of histamine release than peritoneal cells for an equimolar concentration of NT (19.2 2% release for peritoneal mast cells versus 45 + 6% release for pleural mast cells at 10 /xM NT). Moreover, we have also observed anecdotally differences between various populations of mast cells and between various populations of rats in terms of their responsiveness to the same batch of NT. 

The ability of SP to stimulate histamine release from isolated rat peritoneal mast cells is now well demonstrated [31, 97-101], The release is rapid (< 1 min), non-cytotoxic, dependent on a supply of Ca and metabolic energy, and independent of cell-bound IgE [99]. Moreover, as with other peptides, its secretory effect on the mast cell is affected by moderate levels of extracellular cations. For example, the addition of Ca to the bathing medium after the addition of SP increased the secretory response of the cells, while adding calcium (0.1-1 mM), magnesium (1-10 mM) or cobalt (0.01-1 mM) to the cell suspension before SP inhibited histamine release, suggesting the possibility of cation competition for SP binding [99]. 

In humans, the intradermal injection of SP (10 7-10 5 M) produces a flare, wheal and itching, and these effects are prevented by pretreatment with chlorcyclizine (an antihistamine) or by local pretreatment with compound 48/80 [ 103, 104]. It causes histamine release from sections of human foreskin [105]. These authors suggested that SP is significantly less active on isolated rat peritoneal mast cells than in human skin. 

Dynorphin, a-neoendorphin and /1-endorphin each produced a dose-dependent (10 6 M to 10 4 M) release of histamine from rat peritoneal mast cells but not from rat mucosal mast cells which were isolated following collagenase digestion [128]. When administered intradermally to the forearms of human volunteers, dynorphin, /f-endorphin, Leu-enkephalin and morphiceptin each produced a wheal and flare reaction at nM concentrations. Mast cell degranulation was confirmed by electron microscopy of biopsy samples and by its inhibition by hyroxyzine pretreatment [129]. 

Several other cell types have also been shown to secrete histamine-releasing activity, some of which may be peptide in nature (although more work is necessary for a definitive characterization). For example, human lung macrophages cultured for 24 h have been shown to release a soluble factor (12 and 30 kDa) that stimulates isolated human lung mast cells and human basophils to release histamine [ 145]. The generation and release of this factor developed over time (> 1 h) and was blocked by cycloheximide, indicating that protein... 

Bk has been shown to stimulate histamine release from isolated peritoneal rat mast cells [30, 87] and this secretory response, like that elicited by other peptides, requires a source of metabolic energy and is prevented by depletion of cellular Ca [30, 87]. The subsequent treatment of such cellular Ca-depleted mast cells with Bk produces an inactivation or desensitization phenomenon to the subsequent addition of extracellular Ca (secretion declines as the time... 

Figure 4.6. Log dose-response relationships for the effects of synthetic neurotensin, synthetic brady-kinin, and various preparations of NRP on the release of histamine from isolated rat mast cells [73], Each point is the mean obtained for two separate incubations.

Figure 4.7. Histamine release from mast cells in response to various dilutions of HRA generated from bovine serum albumin (BSA) by medium derived from stimulated rat neutrophils [156]. Neutrophils ((50-100) x K lml) were stimulated with FMLP (10 5 M), the medium removed and incubated with BSA (10 mg I ml) at pH 4.5 for 18 h at 37° C. It was then boiled and centrifuged (11,000 x g for 30 s), the supernatant fraction was removed, its pH was adjusted to 7.2 and it was added, at various dilutions, to suspensions of mast cells. Histamine release was then measured after 10 min. As the amount of generated HRA was increased histamine release increased to a maximum at 57 4%. Mean + S.E.M., n = 5. Inset Time-course of generation of HRA as assayed by histamine release from isolated mast cells. HRA was generated as before using 50 x 106 neutrophils/ml. Aliquots were removed at the indicated times and assayed (at 50% dilution) for HRA. Note that there is a significant generation of HRA by 2 h. Mean S.E.M., n = 3.

Evidence for the specific binding of peptides to isolated mast cells is limited. The most detailed study to date has been done by Lazarus et al. [86, 158] who... 

---