This study showed that each type of atypical MC in mastocytosis has its protease profile, which is characterized by the ratio of proteases content within a cell. The total set of MCs determines the integral functional potential of tryptase and chymase proteases in the bone marrow. We have also revealed the expression of nonspecific carboxypeptidases A1, A2, and B in all MCs. Three MC states can be distinguished, depending on the cytotopographic features of the distribution of carboxypeptidases. The presence of carboxypeptidases A1, A2, and B in the cytoplasm of MC indicates actively occurring protein processing processes.
Localization of carboxypeptidases mainly in mature type III granules or immature type II granules indicates the absence of post-translational proteases modification necessary for incorporation into granules.
At the same time, intragranular protease processing continues even when the transcription of tryptase and chymase genes ceases, indicating the possibility of certain rearrangements in the enzyme’s structure and biopolymers localized in secretory granules. The identified feature is of great importance for the realization of the specific proteases functional potential after secretory granules exocytosis into the extracellular matrix, implying the possibility of prolonging the MC secretome regulatory role, including tryptase and chymase. The formation of atypical MC hypogranular forms may be due to the specificity of the mechanisms responsible for secretion into the extracellular matrix.
Additional criteria for mastocytosis: cytotopography of processing and secretion of specific proteases
The pathogenesis of mastocytosis is closely associated with the biogenesis of specific proteases. About 5% of the genome encodes information on human MC proteases, and the protease mRNA content is comparable to the content of housekeeping genes transcripts. This reflects the high content of proteases in the cytoplasm of MC 7,15,35–39.
Tryptase and chymase processing ends with the formation of monomeric or tetrameric forms of enzymes. In particular, the tryptase tetramer with a molecular weight of 140–142 kDa has maximum biological activity and consists of four identical monomers stabilized in the presence of heparin or other glycosaminoglycans at pH values below 6.5 40. The stability of tryptase in МC granules also depends on the histamine content 41. Chymase, in contrast to tryptase, is active in monomeric form and is localized in granules also in combination with heparin. Once in the extracellular environment, the chymase remains in complex with heparin, which protects it from neutralization by endogenous inhibitors 42. At the same time, the chymase activity is known in the absence of heparin, which makes it an understudy to a certain extent of tryptase, ensuring the development of similar physiological and pathological effects in the formation of conditions for a local decrease in glycosaminoglycans in the tissue microenvironment.
MC proteases during the accumulation of granules in the cytoplasm do not show enzymatic activity due to the characteristics of the pH level and interaction with serglycin and glycosaminoglycans 9,12,43. Therefore, an indirect judgment on the content of proteases in MC granules can be obtained after using various metachromatic staining options 26,44–46.
Packaging specific proteases in granules
The classical model of the formation of MC secretory granules suggests that after protease synthesis in rough endoplasmic reticulum they enter the Golgi complex, where they undergo post-translational modification and are packaged in small-sized granular formations surrounded by a membrane 47,48. These granules containing specific and non-specific proteases complex with proteoglycans in the cytoplasm can merge, and after combining with acid hydrolases from type early endosomes, type I secretory granules are formed (Fig. 7)..
Morphologically, they are indistinguishable, small in size, and are further combined with the formation of secretory granules of type II, reaching visualized sizes from 0.2 to 0.4 μm (Fig. 7).. At this stage of maturation, type II secretory granules have a certain secretome composition, including glycosaminoglycans, proteases, lysosomal enzymes, etc. Type II secretory granules are enlarged in size by combining with similar or secretory type I granules (Fig. 6, 7).. Their further maturation leads to a further increase in volume and is accompanied by the formation of type III secretory granules with sizes of 0.4–1 µm with the accumulation of a unique composition of specialized secretome components. At the same time, a broad individuality in the accumulation of certain mediators in the formed granules is possible, the amount of which in each MC can be hundreds, with a significant individuality in the content of proteases (Fig. 7).. Such granules can be tryptase-positive, chymase-positive, as well as with the simultaneous presence of tryptase and chymase (Fig. 7)..
Formation of a hypogranulated TC phenotype
The MC’s hypogranular forms formation reflects the active, unfavorable course of the disease, which is accompanied by a high level of protease synthesis, coupled with active liberation into the extracellular matrix. The formation of immature and mature granules activity can be explained by the stochastic model for MC granule growth and elimination with the direct participation of nano-machines 48). In this case, the homotypic fusion of small granules with each other is possible, leading to the unification of their contents and the outer coating with a common plasma membrane (Fig. 7).. At the same time as shown with confocal microscopy, it is possible to suggest the possibility of unequal granules further merging, which might be a common granule maturation and modification mechanism of the secretome intragranular composition (Fig. 6)..
Ultimately, the content and composition of the granules reflect the cell physiological state, adequate to the conditions of local homeostasis, which can be significantly distorted in pathology. The dependence of the various sizes in MC granules formation is closely related to the needs of a specific tissue microenvironment in one or another secretome component and an integral secretory activity. As a rule, the size of the granules correlates with the duration of their stay in the mast cell cytoplasm. Small granules have a higher rate of post- translational modification of the proteases enclosed in them, are practically not stored, and are characterized by rapid exchange of secretome components in the course of active secretion into the extracellular matrix. Obviously, in the case of hypogranulated MC in mastocytosis, the specific proteases maturation can be completed at the level of type I and II secretory granules with further active secretion into the extracellular matrix, leading to various clinical or organ-specific manifestations (Fig. 6, Fig. 7).. Moreover, in atypical MCs, a certain level of mature secretory granules in the cytoplasm may not be maintained, which is a kind of buffer for an adequate response to the need of the extracellular matrix in a particular secretory component. Further on, when discussing the content of proteases in atypical hypogranulated forms of MC during mastocytosis, one should take into account the fact that young granules may be preferred in secretion over mature ones (Hammel I, Meilijson I, 2015). This fact can serve as an additional explanation for the formation of the hypogranulated MC phenotype in mastocytosis.
Secretory granule as a structural entity of functional activity of MC
The granules structure depends on the degree of maturity, the proteases processing stage and the activity of secretory pathways adequately to the tissue microenvironment challenges.
Therefore, in the morphological aspect, it is important to evaluate the topographic features of the arrangement of enzymes in granules. The study of the localization of secretome components in MC granules in mastocytosis was carried out using electron microscopy 49. In the granules, various contrast-rich objects were identified in the form of twisted plates - “scrolls”, as well as grating and/or lattice-like structures, etc. However, these results cannot provide information on the qualitative composition of the visualized structures 50. In several works with the improved technique of immune-electronic histochemistry, the granules ultrastructure dependence on the presence of proteases was shown; for example, granules with tryptase contained “scrolls” that could overlap each other, and granules with chymase were characterized by the presence of a crystalloid lattice 49,51,52. Tryptase could be co-localized with chymase, carboxypeptidase A and cathepsin G in the same granules 49. However, it should be noted that the solution to the problem of the protease co-localization in granules during electron microscopic examination is very difficult due to the effect of fixing the biomaterial, the histological section plane under study and first, the methodological issues of double immunolabeling.
Our data suggest that contrast-reach formations along the periphery of the granules observed by electron microscopy, as well as possibly scrolls, are a morphological reflection of specific proteases location at these loci (Figs. 5, 6).. An immunomorphological study is a very informative addition to electron microscopy, allowing the detection of fluorochrome-labeled proteases in MC, including inside the granules. Modern advances in confocal microscopy with an ultra-high-resolution option provide unique molecular morphological information about the topography of intragranular co-localization of specific MC proteases (Fig. 6)..
Protease profile of TC in the diagnosis of mastocytosis
The ratio of tryptase-positive and chymase-positive MC in the red bone marrow will be of great importance for the specific tissue microenvironment formation, indicating the vector of functional changes presence at a given time. At the same time, it should be noted that the high variability of the expression of the specific protease within each MC, regardless of its type. The co-localization of tryptase and chymase in the same granules (Fig. 6, Fig.7) emphasized the variability of this criterion depending not only on organ affiliation but also on the development of pathology. Thus, the cytological characteristic of the expression and localization of tryptase and chymase in MC is a separate diagnostic value for assessing the progression of mastocytosis.
Secretory pathways of proteases
Evaluation of the secretory pathways, which exert the physiological effects of tryptase and chymase in the intercellular matrix, has a significant information potential. In detail, these processes are described in our previous works 18,34. In parallel with the secretome maturation, the molecular nano-machines associated with the granules allow precise regulation of the release of the necessary mediators from the granules with further transportation to the cytoplasm and extracellular matrix [Blank U et al, 2014} (Fig. 6, Fig. 7). Unfortunately, microscopy does not allow visualization of some events associated with the hypogranulated forms of atypical TC in mastocytosis, such as transgranulation, microvesicular transport, and exosome formation for the secretion of mediators, etc.
Gradual degranulation or a microvesicular transport provide background (constitutive) secretion of tryptase and chymase into the intercellular space, the intensity, despite its weak severity, is determined by the scale of proteases local participation in the local homeostasis regulation 53,54 (Fig.7).. However, it is obvious that with the development of mastocytosis, this tryptase and chymase secretion mechanism can acquire significant activity, despite the absence of morphological evidence. MCs are known to have the ability, under certain conditions, to accelerate secretion hundreds of times per unit time 48. Vesicles of 30–150 nm in size that are peeling from mature granules undergo after intracellular transport a separate secretion into the extracellular matrix. Gradual degranulation is an important signaling system for the interaction of MCs with each other. Considering the tight MC neighboring to each other in the red bone marrow, as a pathognomonic sign of the disease, we can assume the active piecemeal degranulation participation in the MC intercellular signaling using specific proteases.
Proteases can be secreted into the tissue microenvironment via the “transgranulation” mechanism, during which micro bulging of MC cytolemma is formed at specific loci in contact with other cells (Fig. 7).. For example, this can be observed during the contact of MC with each other, as well as in contact with cells of the fibroblastic differon, endothelium, etc. 10,18,34. Finally, a visually indetectable mechanism of MC proteases secretion into the extracellular matrix is possible through the exosomes formation 55 (Fig. 7)..
Along with the above degranulation mechanisms, there are other options for proteases release into the extracellular matrix, which was observed in granular forms of atypical MC in mastocytosis, as well as in typical MC of red bone marrow. With the kiss-and-run secretion mechanism 53, MC granules came into contact with the plasma membrane to form a temporary pore releasing the proteases into the extracellular matrix in corresponding amount with slightly higher intensity compared to the microvesicular secretion mechanism 53 (Figs. 2C, 2G, 2H-K, 3A, Fig. 7).. The peripheral arrangement of granules in type I MC indicates the active use of this mechanism in specific proteases secretion. The further fate of tryptase and chymase in the extracellular matrix depends on many parameters that determine the rate of protease cleavage from serglycin and subsequent diffusion in the intercellular matrix.
Proteases can enter the extracellular matrix using the mechanism of “macrovesicles” formation, which are fragments of the TC cytoplasm containing mediators that are gradually able to be secreted by other mechanisms and diffuse to the target substrates 34. MC granules or individual fragments of their cytoplasm in the stroma of the organ have autonomy in decision-making and can participate in achieving the required chymase and tryptase concentration within the tissue microenvironment necessary limits without a “maternal” MC participation 34. However, in mastocytosis, this mechanism is quite rare and is a characteristic of predominantly mature MCs.
In the case of allergy, anaphylactic degranulation of MC can be observed. It is accompanied by a massive excretion of granules into the extracellular matrix with a generalization of the process. Perhaps, in some cases, this can significantly aggravate the course of mastocytosis.
Specific МС proteases as multifunctional mediators
Specific proteases bio-effects development in the red bone marrow begins from the moment they enter the extracellular matrix and is characterized by a number of specific features. In particular, specific proteases are involved in collagen fibrillogenesis (Atiakshin D, Buchwalow I, Tiemann M., 2020). This explains the frequent detection of sclerosis and collagen fibrosis in the red bone marrow associated with the prevailing presence of atypical MCs in mastocytosis 56.
Tryptase has a high biological activity, affecting the state of many cellular and non-cellular components of the tissue microenvironment 8,38–40,57. At the same time, secreted MC proteases can lead to further intensification of degranulation using the autocrine mechanism, as well as to increase the liberalization of biogenesis products in eosinophilic granulocytes 38.
Tryptase has its molecular targets on the cells or components of the extracellular matrix, causing any pro- or anti-inflammatory effects 38,42,58–62. Most often, tryptase initiates the development of inflammation, causing an increase in the permeability of the capillary wall, increasing the migration of neutrophils, eosinophils, basophils and monocytes beyond the microvasculature 63. These effects of tryptase can be mediated by the induction of the formation of kinins, IL–1, and IL–8 in the endothelium, which is combined with a change in the synthesis of ICAM–1 intercellular adhesion protein. Several studies have shown the close involvement of tryptase in the processes of angiogenesis. Moreover, the formation of new vessels is combined with the pronounced connective tissue remodeling, associated primarily with the degradation of the extracellular matrix amorphous and fibrous components, the growth factors secretion, cytokines and chemokines, matrix metalloproteinases (MMPs).
The synchronous secretion of MMPs and tryptase may have a reasonable explanation since the latter has the properties to activate various MMPs synthesized not only by MC but also by other cells of the connective tissue in an inactive form within the tissue microenvironment. This list includes MMP–1, MMP–2, MMP–3, MMP–9, MMP–13, etc.
Thus, tryptase, by activating MMP, is capable of exerting far-reaching extracellular matrix rearrangements associated with degradation of both the fibrous component and ground substance components, including laminin, fibronectin, several proteoglycans, etc. 10,18,40,57. Finally, the effects of tryptase on the fibroblastic differone cells are shown, causing their active movement, mitotic division, and stimulation of the collagen proteins synthesis. As a result, the effects of tryptase may promote wound healing and can lead to fibrotic effects10,57,64.
Tryptase has a high tropism for PAR–2 receptors, potentiating the development of inflammation. Localization of these receptors on various cells of a specific tissue microenvironment can lead to pro-inflammatory signaling, including afferent neurons. An important tryptase regulatory mechanism in potentiation of inflammation is the induction of a persistent increase in PAR–2 receptor expression in various connective tissue cells. In particular, in the airways, this leads to the formation of functional prerequisites for exacerbation of bronchoconstriction, mucus secretion by mucocytes. PAR2 promotes M1 macrophage polarization and inflammation via FOXO1 pathway 65. In cells of certain areas of cartilage tissue, PAR–2 receptors increased expression leads to arthritis progression, degradation in osteoarthritic cartilage, inflammation, chondrocyte apoptosis, and cartilage breakdown 66. After surgical interventions, an increase in the presence of PAR–2 on soft tissue cells significantly complicates the course of the postoperative period. It was shown that PAR2 enhanced the expression of MYO10 through the repression of miR–204. PAR2 mediated tryptase-induced cell migration and might contribute to the invasion of cancer cells at the edge of tumor 67. In addition, an important effect of tryptase is the activation of secretion by cells of a specific tissue microenvironment of pro-inflammatory mediators into the intercellular matrix, creating an increased background content of cytokines chemokines68,69.
In light of the previously described effects of tryptase, the progression of allergic reactions is also important 70. Tryptase leads to the stimulation of histamine liberalization from intracellular depots, which, in turn, causes a new increase in tryptase secretion. This contributes to the involvement of new MCs in the degranulation process, creating conditions for the realization of the biological effects of histamine over a larger area 71.
In the literature, various mechanisms of the influence of tryptase are considered that contribute to the growth and differentiation of new blood vessels, including oncogenesis 72,73. The stimulating role of tryptase in neoangiogenesis in B-cell non-Hodgkin lymphoma, multiple myeloma, chronic lymphocytic leukemia, melanoma, etc. has also been shown [Ribatti D. 2016]. Recently, interesting information has appeared about the possible anti- oncogenic mechanisms of tryptase 55.
The substrates of another specific TK protease, chymase, are various components of the extracellular matrix, receptors, proteins, as well as chemokines and cytokines [Pejler G et al., 2007]. Human chymase actively hydrolyzes angiotensin I to its active form of angiotensin II, participating in both local and systemic mechanisms of blood pressure regulation in physiological and pathological conditions, in the pathogenesis of hypertension. In addition to the function of angiotensin II as an effector peptide of the renin-angiotensin system, it also has effects on the regulation of cell growth, angiogenesis, regeneration, and tissue remodeling10,74.
Chymase is capable of causing MC migration to the place of destination in tissues and thus acts as an inducer of directional movement 75. Chymase is involved in the development of pulmonary hypertension and fibrosis. Chymase inhibitors reduced pulmonary hypertension, improved hemodynamics, decreased right ventricular hypertrophy, remodeled blood vessels, and reduced connective tissue in the lungs 76.
Chymase is directly capable of changing the state of many extracellular matrix components and, in comparison with tryptase, has a more pronounced destructive potential 8. On the other hand, chymase is less resistant to inhibition and neutralization by extravascular antipeptidases, including serpins and α2-macroglobulin, and has a shorter interval of enzymatic activity in the tissue microenvironment. Chymase can lead to direct effects of the fibronectin degradation and its fragments accumulation in the intercellular substance of connective tissue, as well as vitronectin, laminin, and other components. The chymase effects can be mediated, in particular, by activation of collagenase, MMP–2, MMP–9, inhibition of TIMP–1, etc. Chymase can induce an increase in mitotic and biosynthetic activity of fibroblasts. An important fact is the participation of chymase in the procollagen molecules enzymatic rearrangement, which makes possible the tropocollagen polymerization with the collagen fibrils and fiber growth formation both in length and thickness 10.
An increase in chymase level correlated with the development of fibrosis in experimental diabetes and autoimmune liver fibrosis, while the use of chymase inhibitors reduced its progression 8. The role of chymase in the formation of keloid skin scars has also been shown 13. Our previous studies convincingly indicated MCs direct participation in fibrillogenesis by using a pronounced inductive effect on the ratio of the tissue microenvironment components, including areas around the fibroblastic differone cell 10. Also, the ability of MC to actively secrete fibrillogenesis inducers is often expressed in the formation of its initiation points close to the MC cytoplasm.
Chymase has a different effect on bioactive peptides. On the one hand, it can activate IL–1β, IL–8, IL–18, neutrophil-activating peptide 2, transforming growth factor-beta, endothelin–1, etc. In this regard, the ability of a chymase, like a tryptase, to provide pro- inflammatory effects causes the recruitment of neutrophils, eosinophils, basophils, monocytes, and lymphocytes into the tissue microenvironment 77. Like tryptase, chymase is actively involved in angiogenesis, which contributes to the progression of cancer 78. On the other hand, it can correct the area of the ischemic lesion, the number, and histoarchitectonics of intraorgan vessels, including the myocardium. Chymase can cause allergic reactions in the skin with the degradation of the structures responsible for attaching the epidermis to the basement membrane in dermatitis, atopic dermatitis and eczema, increase the permeability of the mucous membranes, reduce the epithelium barrier function, increase the vascular wall permeability, the synthesis of IgE and IgG1, inhibit smooth muscle cell proliferation and cause their apoptosis development.
In particular, chymase is involved in the pathogenesis of increased vascular permeability in preeclampsia and Crohn’s disease. Chymase is a powerful MC degranulation inducer and a stimulator of histamine excretion into the extracellular matrix, which leads, to one degree or another, to the generalization of peritonitis, the aortic aneurysm formation, the myocardial region’s expansion with metabolic disorders in heart attack, the secretory activity activation of glandular cells in the respiratory tract, etc. On the other hand, chymase causes degradation of tumor necrosis factor, substance P, VIP, kallikrein, bradykinin, complement component C3a, IL–1β, IL–5, IL–6, IL–13, IL–18, IL–33, preproendothelin –1, tumor necrosis factor-α and eotaxin 77. Reducing the transition processes intensity of cholesterol into macrophage lipoprotein bodies, chymase leads to the formation of “foamy cells” and, accordingly, to the atherosclerosis progression. A close association of the chymase effects with an aortic aneurysm, chronic trophic leg ulcers, lung, liver and kidney diseases, diabetic nephropathy and retinopathy, conjunctival epithelium apoptosis, the development of systemic scleroderma, arthritis, etc. has also been shown 8,79.