Mast cell granules were examined by fully quantitative X-ray microanalysis of 20 cells in freeze-dried cryosections. The mast cells were situated mainly in the connective tissue of the thymic capsule of five adult male Carworth Sprague Europe rats. In addition 30 red blood cells were analysed from the same sections.

Nineteen of the mast cells had granules rich in S and K. One cell had smaller granules, and in this cell the granules contained high [Ca] and [P] instead of high [S] and [K], In the majority of cells (13) the S: K ratio was highly correlated and less than 2–2, whereas in the remaining six cells the individual granule ratios were very variable in any one cell and much higher. The mean granule [K] (994 ± 57 mmol kg-1 dry wt) was about four times the mean cytoplasmic level of 227 ±81 mmol kg-1 dry wt. The existence of this difference in concentration between the granules and the cytoplasm suggests that the K in the granules must be bound.

The relationship between the [K] and [S] is discussed with regard to the possible binding of heparin and amines in the granules.

The granular matrix of mast cells is a dense complex of mainly cationic proteins, acid mucopolysaccharides and bioamines (Padawar, 1979). Heparin, a sulphated polysaccharide, is usually greatly in excess of the amines, which can be histamine, serotonin, dopamine or various combinations of these. Whilst the two can link together to form a salt, the heparinate of histamine, there is usually a great excess of heparin over histamine (West, 1959) and the binding is probably more complex, involving a basic granule protein (Uvnas & Åborg, 1977). Granules isolated in nonionic media retain the size and the electron opacity of the granules in situ, but are said to be unstable in the presence of mineral cations (Na+, K+, Ca2+ and Mg2+), traces of these ions causing catastrophic swelling and an explosive release of amines (Padawar, 1979).

Mast cells are well known to be associated with thymus glands; their presence there has been reported in normal and diseased conditions (Burnet, 1965; Goldstein, 1966). Thymic tissue is a rich source of mast cell precursors (Ginsburg & Sachs, 1963; Csaba et al. 1962; Ginsburg, 1963; Ginsburg & Lagunoff, 1967; Ishizaka et al. 1976) and developing mast cells may be found in the thymus of many animals (see Kendall, 1981). Increases in thymie mast cell numbers have been reported, especially after thymic atrophy (Griss, 1970; Ruitenberg & Elgersma, 1976; Ruitenberg & Buys, 1980; Chatamra et al. 1983). In the continuation of the latter study (Chatamra et al. 1986), the mast cells were noted to be mainly within the connective tissue of the capsule and septa, whilst immature cells were observed within the cortex.

We are currently undertaking a study of different cell types in the rat thymus gland using the technique of X-ray microanalysis. This permits the examination of the elemental content of mast cell granules in vivo, without the disruption of cell fractionation and granule separation techniques. Avoiding such procedures may be important in the study of granule composition, as Jones et al. (1979) have shown that isolating nuclei causes an alteration in their ionic content.

Small pieces (1 mm cubes) of cortex and capsule were excised from the thymus glands of five adult male Carworth Sprague Europe rats (approx. 250g) after anaesthetization with chloroform. The cubes of tissue were placed on small brass stubs and frozen rapidly in just-melting liquid Freon 22 in liquid N 2. The blocks were stored in liquid N 2 before cutting at −65 to −70°C in a Slee cryoultramicrotome. Ribbons of frozen sections approx. 300 nm thick were collected onto Formvar-coated Ni grids and freeze-dried in a stream of liquid N2 in the cabinet at the cutting temperature for approx. 1 h before being placed in a desiccator and left to warm up to room temperature overnight. The dried sections were carbon coated and then analysed in an AEI EMMA-4 fitted with a Link 860 Series 2 energy dispersive detection system. Spectra were collected over 100 s live time with a probe area of 0·8 μm, an accelerating voltage of 60 kV and a 4 nA probe current. A gaseous N2 cold finger was used to reduce contamination. The spectra were processed using the Quantem program (Link Systems Ltd). This program is described by Hall & Gupta (1982). Quantification of spectra was achieved by comparison with previously prepared gelatin standards. Full details of the method and validation of the standards are given by Kendall et al. (1985).

Generally 10 granules were analysed from each of 20 mast cells; the data derived from the analysis of 30 red blood cells are also included here. Student’s t tests were performed where necessary.

The mast cells were mainly located in the capsule around the cortex of the thymus, often quite close to blood capillaries. The red blood cells (RBCs) were situated in these blood capillaries or within the adjacent cortex.

Rat thymic mast cell granules range in size from approximately 0·3 to 1·0 μm in diameter (Fig. 1A), so that the probe used for analysis was generally contained within the granule except where the granules were small, when some cytoplasm was included in the analysis. Owing to the dense packing of the granules (see Fig. 1B) only a very small number of analyses (8) could be obtained from the cytoplasm. This number is too small for statistical purposes.

Fig. 1.

A. Conventional electron micrograph of a mast cell in the capsule of the rat thymus. Tissue fixed (lh at room temperature) in Karnovsky, cut and stained with uranyl acetate, bv, blood vessel nearby; co, collagen. Bar, 1 μm. B. Freeze-dried frozen section (unstained) of a mast cell from a similar situation as in A. co, collagen. Bar, 1 μm.

Fig. 1.

A. Conventional electron micrograph of a mast cell in the capsule of the rat thymus. Tissue fixed (lh at room temperature) in Karnovsky, cut and stained with uranyl acetate, bv, blood vessel nearby; co, collagen. Bar, 1 μm. B. Freeze-dried frozen section (unstained) of a mast cell from a similar situation as in A. co, collagen. Bar, 1 μm.

Fig. 2 is a typical spectrum from a mast cell granule; the granules in 19 of the 20 cells had a similar composition. The mean elemental concentrations from 158 granules analysed from these 19 cells are given in Table 1, along with the values for mast cell cytoplasm and RBCs from the same tissues. When these are compared with the results of the analysis of RBCs and the few mast-cell cytoplasm estimates it can be seen that the granules contain much higher [S] and [K], and possibly raised [Na] and [Zn],

Table 1.

Mean f±S.Ej concentrations of elements (mmol kg −1 dry wt) in red blood cells, in mast cells and from one cell with granules high in Ca and P

Mean f±S.Ej concentrations of elements (mmol kg −1 dry wt) in red blood cells, in mast cells and from one cell with granules high in Ca and P
Mean f±S.Ej concentrations of elements (mmol kg −1 dry wt) in red blood cells, in mast cells and from one cell with granules high in Ca and P
Fig. 2.

A typical spectrum obtained from the analysis of a freeze-dried frozen section of a mast cell granule.

Fig. 2.

A typical spectrum obtained from the analysis of a freeze-dried frozen section of a mast cell granule.

The ratio of S: K in each granule was calculated and the mean ratio was found to be 3·0 ±3·7 but as the distribution of the frequencies is skewed (Fig. 3) the median value is 1·4 and 75 % of the granules had an S: K ratio of less than the mean, with the maximum frequency at 0·8—0·9 (22%).

Fig. 3.

The frequency distribution of the ratios of S:K in individual mast cell granules (n = 158) from 19 cells.

Fig. 3.

The frequency distribution of the ratios of S:K in individual mast cell granules (n = 158) from 19 cells.

The mean ratio of S: K was calculated for each cell and found to be significantly correlated in 13 cells. The individual granule values of [S] and [K] in these cells are indicated by dark circles in Fig. 4. In 11 cells the correlation coefficient (r) is 0·9 or 1·0 (P< 0·001) and in two cells 0·7 (P< 0·1). The ratio of S: K in all of these cells is less than 2–2.

Fig. 4.

The individual granule [S] and [K] in 19 cells. (○) Granules from 6 cells where the cell [S] and [K] were not correlated. (•) The others show a high correlation (coefficient = 0·74; P< 0·001; Y = 411+0·828×1).

Fig. 4.

The individual granule [S] and [K] in 19 cells. (○) Granules from 6 cells where the cell [S] and [K] were not correlated. (•) The others show a high correlation (coefficient = 0·74; P< 0·001; Y = 411+0·828×1).

The cells with ratios over 2·2 were six cells in which there was no correlation (r=<0·7) between [S] and [K]. Individual granules from these six cells are indicated in Fig. 4 by open circles. Most of the granules had low [K] but a few had higher [K], similar to granules from the remaining 13 cells in which the [K] and [S] were higher correlated (filled circles, Fig. 4). The six cells with‘high ratios’ (mean 6·9 ± 4·7) came from two animals, and one cell was not situated in the capsule with the rest, but within the cortex. This cell appeared morphologically immature, as judged by the irregular size and variation in electron density of the granules, and the presence of more cytoplasm between granules. This probably corresponds to a stage II mast cell (Combs, 1966).

The mean elemental concentrations of the granules from these two populations of mast cell (designated‘low’ and‘high’ ratios) are shown in Table 2. The high-ratio granules had less Na, P, Cl and K (P< 0·001), and more Zn (P<0·01) and Ca (P<0’001), than the low-ratio granules. There was no difference in the [Mg], [S] and [Fe] or in the background integrals (low-ratio mean = 6556 ± 230 compared with 5880 ± 253 for high-ratio granules).

Table 2.

Mean (±S:E.) concentrations of elements (mmolkg −1dry wt) in mast cell granules with low (<2·2) and high (>2·2) StKratios

Mean (±S:E.) concentrations of elements (mmolkg −1dry wt) in mast cell granules with low (<2·2) and high (>2·2) StKratios
Mean (±S:E.) concentrations of elements (mmolkg −1dry wt) in mast cell granules with low (<2·2) and high (>2·2) StKratios

In one of the 20 cells, the granules (see Table 1) were rich in Ca and P, very low in S and had low [K] (S: K ratio 0·3). The granules appeared to be slightly smaller than those of the mast cells discussed above.

Altogether 20 cells were studied by X-ray microanalysis, and in all but one of these cells the spectra obtained from the granules were broadly comparable in their very high S and K contents. A high [S] in mast cell granules is predictable, as the granules contain much heparin, but the high K levels were unexpected. One cell was completely different (high [Ca] and [P]). Either this cell is not a mast cell (its granules were smaller) or this mast cell contains little or no heparin. The significance of the findings in this cell is not known.

Padawar (1979) reported that mast cell granules isolated in non-ionic media are very unstable, small traces of the common mineral cations Na+, K+, Ca2+ and Mg2+ causing swelling (up to 30 times) and an explosive release of bound amines. A previous X-ray microanalysis study of mast cell granules did not mention K (Yarom et al. 1975), but in that work the results referred to eight granules analysed from conventionally fixed, dehydrated and Epon-embedded electron-microscope (EM) material. A lack of K is not surprising as the preparative procedures would have caused the loss of any readily diffusible elements, and because of this Na and K are not generally detectable in conventional EM embedded specimens.

A study of the S:K ratios within individual granules revealed a much more complicated picture than the mean values suggested. The mode of this skewed distribution was about 1–0, but granule ratios in six cells were much higher and very variable. Since one of these cells appeared immature, the variation may have developmental significance, so it is important to know whether these higher and variable ratios have a biological basis or are a consequence of the analytical technique.

The quantitative procedure used here relies upon the continuum normalization method of Hall as described by Hall & Gupta (1982). Major factors that affect the reliability of the method are contamination and mass loss (Hall, 1979; Hall & Gupta, 1983). Contamination could cause deposition of S, thus affecting quantification of the elements, but there was no evidence of that on the films. Mass loss from the organic matrix of specimens is now well documented (Hall & Gupta, 1974; Rick et al. 1981; Andrews et al. 1981) and is generally considered to be complete well below the beam doses that we have used. Mass loss from the organic matrix would affect quantification of all elements, and elemental loss from the S Kαpeak, which is known to be sensitive to beam damage, would affect ratios. Differences in mass loss between the mast cell granule matrix and the protein matrix of our standards would cause errors in absolute quantification.

Mast cell granules contain basic protein, amines and heparin, a sulphated polysaccharide from which S can be lost at the probe current used. In addition, Shuman et al. (1976) showed that the mass loss from a sucrose specimen was greater than that from serum albumin (a protein), so mass loss from the polysaccharide might also add to problems in quantification. However, comparisons between high- and low-ratio granules should be valid, as all mast cell granules presumably have a similar composition in terms of organic matrix. Thus it might be expected that behaviour under the beam would be similar in both groups. It is notable that under the operating conditions defined here, there was no significant difference between the background integrals from the low- and high-ratio granules, suggesting that quantification was not affected differently in the two groups of cells. It should also be noted that the mean S content in these two groups of granules is similar, with the variation in the S: K ratio being caused mainly by variation in the K content, and K is generally considered stable under the electron beam. Thus it can be concluded that the variation in S:K ratios observed probably has a biological basis.

The few mast cell cytoplasmic [K] estimations recorded adjacent to granules were considerably lower than the granule values. More analyses of cytoplasm are needed for confidence in these values. Work on dog pancreatic exocrine cells (Nakagaki et al. 1984) has shown that the water contents of cytoplasm and granules in a cell are not the same. If this is also true for mast cells then values expressed in terms of dry weight might be expected to differ. Nakagaki et al. (1984) found that the dry mass fraction of pancreatic acinar cytoplasm was 22% and that of the zymogen granules 37 %. Those values were similar to the results of their previous work on dog salivary gland cells (Sasaki et al. 1983). Allowing for water contents of this order, the mean [K] in the low-ratio granules was about 10 times the cytoplasmic level and one or two granules had approximately 27 times more K. The mean high-ratio granule [K] was three times that of the cytoplasm. This means that K must be bound in the granules.

In the studies reported by Nakagaki et al. (1984) the [K] in the granules was lower than that in the surrounding cytoplasm, in contrast to our findings here. Roomans & Wei (1985) also found that rat pancreas zymogen granules were low in K. Pancreatic granules are largely proteinaceous, unlike mast cell granules, which are sulphated carbohydrates. In other microprobe studies of tissues producing carbohydrate secretions high [K] are recorded. Although the work was not fully quantitative, Appleton et al. (1979) found elevated [K] close to the mucus surface of the mantle collar epithelium in a snail (Otala lactea) and the microprobe studies from the laboratory of Hall and Gupta have shown that sites rich in sulphated glycosaminoglycans sequester K (and Ca) from the surrounding medium (see references quoted by Gupta & Hall, 1981). More recently their studies have considered the mechanisms involved in producing and maintaining such high K concentrations (Dow et al. 1984; Gupta et al. 1985). Also, Scott (1978) considered the preferences of various polyanions for Na and K, and found that ester sulphates strongly prefer K. Thus there is a precedent for high [K] in some biological situations.

Although it was once thought that Zn assisted in the binding of histamine in the granule by a chelating action, Bergqvist et al. (1971), after recording 16mmol Znmg’1 dried granules, considered this amount too low to explain such an effect, and thought it more likely that the Zn originates from granule enzymes. The mean Zn concentration recorded in this study (10 ± 1 mmol kg-1 dry wt) is slightly lower than that of Bergqvist et al. (1971), but the group of‘high S: K ratio’ granules had a mean value of 14 ± 2 mmol kg-1 dry wt, which is quite a close agreement.

If the cells with high and variable granule ratios are immature, then immature granules may have a wide variation in their K content, and it appears that with maturity the S: K ratio approaches 1·0. This narrow range of ratios in the majority of granules implies a functional role for K in binding anions.

Until the identification of a basic granule protein (Lagunoff et al. 1964; Bergqvist et al. 1971) it was generally assumed that heparin was bound to histamine or other amines in the granule. The heparin-histamine complex is a stable salt, binding efficiently at pH 2–3, but at neutrality (probably nearer to the physiological pH) the binding capacity of heparin would account for only about 10% of the histamine. With a low molecular weight basic protein binding to heparin the terminal COO-group becomes the only group available for cation binding (Uvnäs et al. 1970; Uvnas & Aborg, 1977). Thus a ratio of S:cation should be 2:1 (or 0·5). This low ratio was not observed in terms of S: K in any granule and although the inclusion of other cations would improve the ratio, their contribution is not likely to be substantial.

A direct binding of K to heparin would give an S: K ratio of 1·5, but leave the roles of the protein and histamine unaccounted for. However, although ratios around 1–2 are commonly found in these studies, there is much variation from cell to cell in the observed granule ratios, and it is conceivable that there are a number of different possible ways in which the heparin, granule protein and histamine (or other amines) are arranged. Thus small changes in the relative proportions of any component - the production of more amines, for example - might then alter the binding patterns. This view would be in accord with the interpretation of the apparent increased sulphation of mast cell granules with age (increased carbohydrate moiety seen with dye reactions) as being caused by changes in dye-binding sites. A small measure of support for this is given by the similar mean S concentrations in the granules of highland low-ratio cells.

Finally, although granules isolated in non-ionic media appear to look like granules in vivo, this study shows that there could be differences in their composition. A loss of K in the isolation procedures would alter the binding of granule components and perhaps account for the instability of mast cell granules in the presence of cations after isolation.

This study prompts a new appraisal of heparin and histamine binding in the mast cell granules to take account of the high [K] observed. Some thoughts on the effect of releasing high K levels at degranulation are also necessary as the membrane permeability of surrounding cells could be greatly influenced by this event, if only for a brief period of time.

We thank the Sir Jules Thorn Charitable Trust for financial support to work on the thymus gland, and Mr Ian W. Morris for technical assistance.

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