Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2K ^b mutants analyzed.Abbreviations used in this paper B6 C57BL/6Kh - bm5 B6-H-2^bm5 - bm6 B6-H-2 ^bm6 - bm7 B6.C-H-2 ^bm7 - bm9 B6.C-H-2 ^bm9 - bm16 B6-H-2 ^bm16 - D H-2D - K H-2K - MHC major histocompatibility complex     

An histochemical approach to characterization of anionic constituents in mast cell secretory granules

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO₄ and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500–900 gold particles/m²), while in other peritoneal cell granules it was about 10 times less (ca. 40–80 gold particles/m²). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.     

Regulation of secretory granule size by the precise generation and fusion of unit granules

Morphometric evidence derived from studies of mast cells, pancreatic acinar cells and other cell types supports a model in which the post-Golgi processes that generate mature secretory granules can be resolved into three steps: (1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; (2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule’s volume, as well as changes in the appearance of its content and (3) fusion of secretory granules of the smallest size, termed ‘unit granules’, forming granules whose volumes are multiples of the unit granule’s volume. Mutations which perturb this process can cause significant pathology. For example, Chediak–Higashi syndrome / lysosomal trafficking regulator (CHS)/(Lyst) mutations result in giant secretory granules in a number of cell types in human beings with the Chediak–Higashi syndrome and in ‘beige’ (Lyst^bg/Lyst^bg) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule–granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules permit the cells to store large amounts of material without requiring the amount of membrane necessary to package the same amount of cargo into small granules. In addition, the formation of mature secretory granules that are multimers of unit granules provides a mechanism for mixing in large granules the contents of unit granules which differ in their content of cargo.     

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

The histochemistry of complex carbohydrates in certain organs of homozygous brachymorphic (bm/bm) mice

Summary Homozygous brachymorphic (bm/bm) mice are characterized by disproportionately short stature and by undersulphated proteochondroitin sulphate in cartilage with a defect in the synthesis of the sulphate donor (3-phosphoadenosine-5-phosphosulphate: PAPS). In adult mice of this mutant strain, several organs have been examined by light microscopical methods of complex carbohydrate histochemistry, in comparison with those from control (C57BL/6J) mice of comparable ages. The organs included the trachea, aorta, skin, stomach colon and cornea. In the nutant mice, connective tissue elements and components of epithelial tissues exhibited apparently weaker positive reactions for ester sulphate groupings of complex carbohydrates, as compared with those in the control mice. From these results, it is concluded that the undersulphation in complex carbohydrates is widespread throughout the connective and epithelial tissues of the mutant mice.     

An unexpected role for autophagy in degranulation of mast cells

Mast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7-deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.     

Conjugated avidin binds to mast cell granules

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.     

Brown‐midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene

Brown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.     

Mast cells in human bile duct obstruction

Surgical biopsy specimens obtained from 50 patients with secondary cholangitis caused by obstruction of the common bile duct were studied immunohistochemically. Data on the number and ultrastructural appearances of mast cells positive for tryptase, chymase, vasointestinal polypeptide (VIP), and substance P (SP) were obtained. The bile ducts from patients presenting combined chronic exacerbated cholangitis and chronic sclerotic cholangitis showed significantly higher numbers of mast cell types compared to the controls (P < 0.0001). Cases with sclerotic cholangitis alone had significantly lower number of cells than patients with chronic exacerbated cholangitis alone (P 0.0001). Morphometric measurements of electron micrographs showed that mast cell granules containing VIP, SP and chymase were commensurable in size. Electron-lucent granules without reaction product (altered granules) and granules with focal distribution of the reaction product were observed in all types of mast cells. Furthermore, some nerve fibers positive for SP and VIP and serotonin-positive endocrine cells were observed in close proximity to the mast cells. In conclusion, the results of our study demonstrate the existence of different populations of mast cells, nerve structures and endocrine cells in the lower part of the human large bile duct, and suggest their participation in the development of pathological processes.     

Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation

Accumulating in vitro and in vivo studies have proposed a role for mast cells in the pathogenesis of atherosclerosis. Here, we studied the role of mast cells in lipoprotein metabolism, a key element in the atherosclerotic disease. Male mice deficient in low‐density lipoprotein receptors and mast cells on a Western diet for 26 weeks had significantly less atherosclerotic changes both in aortic sinus (55%, P = 0.0009) and in aorta (31%, P = 0.049), as compared to mast cell‐competent littermates. Mast cell‐deficient female mice had significantly less atherosclerotic changes in aortic sinus (43%, P = 0.011). Furthermore, we found a significant positive correlation between the extent of atherosclerosis and the number of adventitial/perivascular mast cells in aortic sinus of mast cell‐competent mice (r = 0.615, P = 0.015). Serum cholesterol and triglyceride levels were significantly lower in both male (63%, P = 0.0005 and 57%, P = 0.004) and female (73%, P = 0.00009 and 54%, P = 0.007) mast cell‐deficient mice, with a concomitant decrease in atherogenic apoB‐containing particles and serum preβ‐high‐density lipoprotein and phospholipid transfer protein activity in both male (69% and 24%) and female (74% and 54%) mast cell‐deficient mice. Serum soluble intercellular adhesion molecule was decreased in both male (32%, P = 0.004) and female (28%, P = 0.003) mast cell‐deficient mice, whereas serum amyloid A was similar between mast cell‐deficient and competent mice. In conclusion, mast cells participate in the pathogenesis of atherosclerosis in ldlr^?/? mice by inducing both an atherogenic lipid profile and vascular inflammation. J. Cell. Biochem. 109: 615–623, 2010. © 2009 Wiley‐Liss, Inc.     

A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.     

Recycling of mast cells following degranulation in vitro: An ultrastructural study

Mature mast cells, isolated from the rat peritoneal cavity, were placed into suspension culture, either as resting cells or after degranulation by exposure to compound $\text{48}\text{80}$, and were maintained for up to 63 hr. No mitotic cells were observed, and cell number was conserved. The culture conditions did not cause spontaneous degranulation and cell survival was better than 80%. However, with time in culture, an increasing percentage of cells acquired a vesiculated appearance, characterized by a Golgi area with distended cisternae, the accumulation of lysosomal or autophagic-like vesicles, and enlarged, irregular or fused secretory granules. In the degranulated group, about one-fourth of the cells recovered the morphological appearance of resting cells by 63 hr, indicating that they are capable of ‘recycling’. A cell type with a unique morphology, characterized by a large central vacuole containing secretory product, an eccentric nucleus, and mature secretory granules at the cell periphery appeared in the stimulated group after 22 hr of culture. It may be a possible intermediate stage in the mast cell regranulation process, based on its occurrence exclusively in the stimulated group, the correlation between its distribution and the recovery of mast cells to the resting state, and the morphological resemblance of its granule contents to stages in granule maturation in differentiating embryonic mast cells.     

The definition of a new IA antigenic specificity using the B6.C-H-2 bm12 I-region mutant strain

A new Ia specificity has been defined using theIA-subregion mutant B6.C-H-2 ^bm12. The immunization to produce the antiserum wasbm12 anti-A.BY, as all other immunizations, such asbm12 anti-C57BL/6, failed to produce antibody. By selecting strains of C57BL origin for testing, it was shown that, (a) the serum was only weakly cytotoxic but gave substantial reactions using a rosetting assay; (b) the antibody reacted with B cells and not T cells; (c) strains of theb, d, p andq H–2 haplotypes were positive, whereasf, k, r ands were negative; (d) absorption studies demonstrated only a single specificity to be present and by testing recombinant strains, the reaction mapped to theIA subregion; (e) SDS-PAGE demonstrated that the antiserum reacted with a molecule of MW 33 000. Preliminary studies indicate this new specificity, present on C57BL/6 and lost frombm12, is present on the same molecule as other I-A specificities.This work was supported by funds obtained from the N. H. and M. R. C. (Australia) and the National Institutes of Health, Grant No. CA-21224.     

Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody

Dense-core granules represent an adaptation of specialized secretory cell to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tet-rahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants. © 1992 Wiley-Liss, Inc.     

Polyamines are present in mast cell secretory granules and are important for granule homeostasis

Background

Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules.

Methodology/Principal Findings

Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system.

Conclusions/Significance

Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.     

In situ characterization of mast cells in the frog Rana esculenta

The number, distribution, and ultrastructural characteristics of mast cells were assessed in the tongue, heart, and kidney of the frog Rana esculenta. The density of tongue mast cells (253±45 mast cells/mm²) was significantly higher than that of the heart (5.3±0.4/mm²) and kidney (15.3±1.4 /mm²). A striking feature of this study was the remarkable association of frog mast cells to nerves. The ultrastructural study of the mast cell/nerve association demonstrated that mast cells were closely apposed to or even embedded in nerves. Mast cells were also physically associated with melanocytes even in the heart. Mast cells were Alcian blue⁺/safranin⁺ in the tongue and in the peritoneum, whereas in the heart and in the kidney they were Alcian blue^–/safranin⁺. The mast cells in the lamina propria of the gastrointestinal tract were Alcian blue⁺/safranin^–. The cytoplasm of frog mast cells was packed with numerous heterogeneous, membrane-bound granules. The ultrastructure of these cytoplasmic granules was unique, being totally unlike any other previously described granules in other animal species as well as in man. The histamine content/frog mast cell (≈0.1 pg/cell) was approximately 30 times lower than that of human mast cells isolated from different tissues (≈3 pg/cell). A monoclonal anti-histamine antibody was used to confirm the ultrastructural localization of histamine in secretory granules in frog mast cells.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.