Ultrastructural Morphometry of Isolated Cells Methods, Models and Applications

Path. Res. Pract, 166,239-259 (1980)

Review

Department of Human Biology and Anatomy, The University, Sheffield, UK

Ultrastructural Morphometry of Isolated Cells: Methods, Models and Applications

T. M. MAYHEW and F. H. WHITE

Summary Isolated cells constitute a favourable experimental model for the pathologist interested in ultrastructural morphometry. A particular advantage is that the same sampling regime may be appropriate for both normal and pathologically-altered material. Often this is not the case with other model systems. This report reviews alternative methodologies for analysing isolated cells, including free cells and dissociated cells in culture. We begin by attempting to define problems of more general concern: preparative techniques, methods of sampling, selection of stereological parameters and possible sources of error. A generalised morphometric model for isolated cells is followed by descriptions of models devised for specific cell types, notably mononuclear phagocytes and cells of the lymphocyte family. Typical results obtained by stereological analyses of these models are presented.

1. Introduction Stereological techniques have been used to characterise many cell and tissue types under normal, experimental and pathological conditions (see Reith et aI., 1976; Rohr et aI., 1976 for examples). However, there have been few attempts to develop detailed model systems suitable for the quantitative ultrastructural evaluation of isolated cells. For present purposes, the term "isolated cell" embraces three main groups: (I) free-floating cells in fluid milieux (e.g., blood leukocytes, alveolar macrophages, cells in serous exudates), (2) cells found in more highly-organised tissues yet retaining a distinct individuality (e.g., thymocytes, histiocytes, lymph node lymphocytes, connective tissue fibroblasts) and (3) cells from a variety of sites which have been dissociated for tissue culture. Isolated cells offer certain advantages as experimental tools for studying aspects of cellular biology. They are often readily available in large

240 . T. M. Mayhew and F. H. White

numbers and as reasonably pure populations showing little morphological variation. Indeed, they have been employed to investigate many phenomena including cytopoiesis, cell differentiation and maturation as well as intracellular events which accompany endocytosis, movement and organellogenesis. Isolated cells are also convenient models with which to study processes of pathobiological interest such as blast transformation and macrophage activation. The cells also have advantages of especial interest to morphometrists. Firstly, stereological data for populations of isolated cells can be correlated with parallel cytochemical, biochemical and physiological data (e.g., Hofer et al., 1972; Helander and Bloom, 1974; Fossum and Gautvik, 1977) so allowing us to define the "average cell". In this way, more objective comparisons of structural and functional changes may be drawn. Clearly, the validity of this sort of study will be influenced by the degree of purity of the cell preparations. Of more immediate practical significance is the fact that isolated cells are relatively easy to sample in a representative manner. This follows by virtue of their very independence of one another. As populations they are not usually arranged in ways which impart structural anisotropy and, even in the event of preferential distribution (for instance, gradients of cell size within a centrifuge pellet), it is seldom difficult to cater for structural inhomogeneity. This often makes it easier to sample normal and pathological cells in the same way. This report presents a selection of techniques suitable for sampling and analysing populations of isolated cells from various sources. We begin by considering problems of general concern relevant to different sorts of isolated cell. We next consider morphometric models developed for specific cells. Inevitably, these have been chosen in a subjective manner as it is not feasible to furnish a comprehensive review. However, they do provide a guide to which readers can relate their particular experimental models.

2. Methods 2.1.

General Considerations

The procedures adopted for stereological analysis are influenced greatly by the structure being studied and, quite often, a structure-specific sampling regime and morphometric model must be devised. For isolated cells the basic procedures are broadly similar so it is possible to present a generalised plan which is, to a great extent, independent of cell type, 2.2.

Sampling procedures

As for any other cell or tissue, rigorous multistage sampling must be

Morphometry of Isolated Cells .

241

performed to ensure a random and representative sample (\Veibel, 1969, 1970). In general, this will be achieved by selecting experimental animals, blocks, sections and microscopic fields without preceding knowledge of their quality and content. An exception to this, involving conscious selection of cells sectioned through their nuclei, is discussed below. Sampling pellets of cells. Most sampling procedures described in the literature are designed to deal with intact tissues which, as fixed cubes or slices, are processed directly for electron microscopy. However, isolated cells often have to be harvested for electron microscopy by sedimentation and the distribution of cells in the resulting centrifuge pellet may not be homogeneous (there may, for instance, be a differential distribution dependent upon cell size). This makes representative sampling a difficult process. Various procedures help one to overcome this difficulty: firstly, pellet homogeneity may be assessed by comparing "vertical" and "horizontal" sections through a given pellet block (Petrzilka et al., 1978). Alternatively, the pellet may be broken into smaller pieces prior to embedding, the resulting blocks being sampled individually. A variation of the latter method applied to cultured cells is described by Steinman et al. (1976): fixed cells are scraped from monolayer cultures and processed as cell pellets which are then divided into small pieces (see also Hirsch and Fedorko, 1968). Davies et aI. (1977) avoid the dangers of pellet inhomogeneity by harvesting fixed alveolar macrophages as pellicles on Millipore filters. In an attempt to preserve representative sampling, pelJicles are subsequently chosen at random and cut to provide blocks from both periphery and centre. This filtration method was originally developed for examining subcellular fractions (Baudhuin et al., 1967). When using cell pellets it may also be necessary to check that cell densities are similar. Differences in the packing of cells may alter their morphology, for example the prominence of cell surface features (Dumont, 1969). Multistage sampling of micrographic fields. The dimensions of cellular structures vary widely (e.g., ribosomes 15-20 nm in diameter; cells 5-15 urn in diameter). For stereological characterisation of different subcellular components, it is therefore necessary to select levels of magnification appropriate to each specific morphometric model. Each level then equates with a sampling stage. The first stage (stage I) usually corresponds to a low magnification sampling which focusses on gross cellular features visible by light or electron microscopy. It may be carried out on smears or spotted preparations of cells, on paraffin- or resin-embedded sections, the final choice being determined by the requirements of the investigation.

242 . T. M. Mayhew and F. H. White

Higher sampling stages (II, III and so on) usually equate with higher magnifications under the electron microscope and seek to evaluate progressively finer details of cell structure. Unbia sed and nuclear-biased sampling. The majority of stereological analyses involve cells and tissues which are readily identified by virtue of their morphological appearance, histochemical properties or diagnostic location. When other criteria are lacking, it is necessary to classify cell type on the basis of morphology alone. This is often the case for populations of isolated cells. A cell pellet, for instance, exhibits no characteristic histological arrangement such as that seen in the liver or the adrenal glands. In relatively pure populations of isolated cells, most random section planes would permit unequivocal recognition of cell type (e.g., Petrzilka et aL, 1978; Woodward, 1978). The same may be true for cells like the mast cell which have characteristic intracytoplasmic inclusions. A selection of such sections would constitute an unbia sed sample of cells. However, the experience of many workers has shown that in mixed cell populations, individuals sectioned through unfavourable planes can be difficult to classify unambiguously. For instance, a lymphocyte might be mistakenly identified as an eosinophil or mononuclear phagocyte (Zucker-Franklin et al., 1966; Davies et al., 1977). To circumvent this problem, it is sometimes expedient to select only cell profiles transected through the nucleus. This conscious selection may be termed nuclear-biased sampling and it often facilitates identification. The approach has been invoked to analyse many isolated cells, including lymphocytes (Konwinski and Kozlowski, 1972; Le Bouteiller et aL, 1976; James, 1978; Al-Hamdani et aL, 1979a), macrophages (Mayhew and Williams, 1971; Steinman et aL, 1976; Davies et al., 1977), plasma cells (Jones and Williams, 1972) and mast cells (Helander and Bloom, 1974)· With this mode of sampling, only nucleate cell profiles are measured . Any other profiles or profile fragments obtruding into the microg raphic field are ignored (Mayhew and Williams, 1971). Whilst unbiased samples are ultimately representative, nuclear-biasing leads to an overestimation of nuclear volume density which must be corrected. Stereological correction procedures are available in the event (Konwinski and Kozlowski, 1972; Mayhew and Cruz, 1973; Cruz Orive, 197 6a). Procedures are also available to correct cell surface/volume ratios (Cruz Orive, 1976b), but they may not always be necessary (Fritsch, 197 1; Mayhew and Williams, 1971; Mayhew, 1979a). Nuclear-biasing may not affect the volume densities of other subcellular compartments, provided they can be referred to the cytoplasm and not whole cell as containing volume (Mayhew and Williams, 1971; Konwinski, personal communica-

Morphometry of Isolated Cells . 243

tion), The same is probably true for surface and numerical densities. 2.3. Generalised morphometric model

Apart from components peculiar to a given type of cell (e.g., specific granules of the eosinophil, basophil and mast cell), the functional compartments of isolated cells are composed of organelles encountered in other cell models. A generalised morphometric model for isolated cells might therefore be represented as in Figure 1. This model is an example and is not intended to be a complete hierarchical division into all possible compartments. It is important to precede quantitative analysis by a thorough qualitative description of cell composition. It is therefore necessary to define each compartment carefully: for example, does the term "rough endoplasmic reticulum" include the outer, ribosome-studded membrane of the nuclear envelope? Does "inner mitochondrial membrane" include cristae membranes as well as the inner boundary membrane? The compartment referred to as "remaining cytoplasm" (or "residual cytoplasm") also varies greatly from study to study (cf., Mayhew and Williams, 1974a; Davies et al., 1977). The tertiary morphometric parameters given in Figure I are those chosen most commonly for correlating cell structure and function. As we shall see, the surface features of isolated cells may be of greater interest than for other cell types. For estimating tertiary data (vide infra), two reference volumes can be envisaged: the volume of the average cell (\Teen) or that of the average nucleus (V N ) . 2.4. Primary and secondary parameters Planar morphometric data gleaned by measuring thin sections with the aid of test lattices are referred to as primary parameters (e.g., point and intersection counts, profile numbers and areas, boundary trace lengths). They are converted to secondary parameters by means of the appropriate stereological relationships which are described elsewhere (Weibel, 1969; Elias et al., 1971). Since profiles of isolated cells vary in area from micrograph to micrograph, it is particularly important to apply these relationships in the correct form in order to avoid statistical bias (Mayhew and Cruz Orive, 1974). The secondary parameters include the so-called component densities in a volume: volume density (V v), surface density (Sv), length density (My) and numerical density (No). With many isolated cells, the cell surface is of especial interest. That of macrophages, for instance, is thrown into finger- and flap-like processes and peripheral invaginations (Williams and Mayhew, 1973; Mayhew and

244 . T . M. Ma yhew and F. H. White

Compartments

Cytoplasm Mitoch ondria Lyso somes Rough ER Smooth ER Golgi clem ent s Ribosomes (free /bound ) Filaments/ tubules Specific incl usions "Remaining cytoplasm"

Tertiary P ar ameters Volume

Surface

V eell

Seell

N eell

VN V eue V het V nue

S!\'

NN

Veyt V mit VJ,.s

v.; v.;

V Ge

Vfil V ine V reyt

Number

Len gth

N nlle Smit Slys Srer Sser SGe

N mit N 1Yb

N Ge N rib Mfil

Sine

N ine

Fig. 1. Generalised morphometric model for isolated cells. ("Specific inclusions" might be eosinophi l or mast cell gra nu les, ph agosomes and so on ).

Williams, 1974a) involved intimately in endocytosis (Steinman et al., 1976; Oakl ey and Mayhew, 1978). This surface also bears specific receptor sites (Wollweber and Fritsch, 1975). The surface may also be of diagnostic value, as in hairy-cell leukaemias (James, 1978). Therefore, stereology of isolated cells often places additional emphasis on secondary parameters related to the plasmalemma surface. One such parameter is volume/surface ratio which relates the volume of a structure to its own surface area (Chalkley et a1., 1949). Recently, other possibilities have been advanced (Mayhew, 1979b) and it is now practicable to estimate component densities on a surface: surface density (58), length density (M s) and numerical density (N«). These permit the estimation of such' information as number of microvilli per unit surface, length of flaplike processes per surface and relative surface of cell membrane occupied by an immunocytochemical marker. They could also be used for counting nuclear pores and surface features (desmosomes, synapses) on other types of cell. Man y parameters ha ve been used to characterise the extent and irregularity of cell and nuclear membranes, including indices of nuclear indentation (Schrek, 1972; Lito vitz and Lutzner, 1974) and cell surface ampli-

Morphometry of Isolated Cells . 245

fication (Williams and Mayhew, 1973; Mayhew, 1979C; Al-Hamdani et al., 1979a). The cell surface amplification factor provides a useful index of how much more surface a cell has than a sphere of equivalent volume. An interesting procedure originally designed to assess the surface features of isolated thyroid cells is described in Vassart et al. (1971). When dealing with cell surface features such as microvillous processes it is important to consider the effects of fixation. The type of fixation may influence not only the number and size of microvilli but also the occurrence of bleb-like extensions of the plasmalemma (e.g., Daems and Brederoo, 1973). 2.5. Tertiary parameters

If secondary parameters can be converted into absolute volumes, surfaces, lengths and numbers, tertiary parameters are obtained. In most cases, conversion requires estimation of a reference containing volume. Usually either the cell or the nucleus is chosen, depending to some extent on which is the more regular (i.e., more nearly spherical). Mean cell volume may be estimated with the aid of a Coulter counter (Heiniger et al., 1967; Petrzilka et al., 1978). A variety of additional procedures is available for sizing cells and nuclei (Heiniger et al., 1967; Mayhew and Williams, 1974aj Woodward, 1978j Petrzilka et al., 1978; Oakley and Mayhew, 1978; Mayhew, 1979C; Al-Hamdani et al., 1979a). While the methods can be used with light and electron microscopy most rely on the assumption that cells and nuclei are spherical and this will introduce an unknown degree of error. With tertiary data, morphological information can be used to describe the average cell in a population and this is a valuable approach for correlating morphometric data with biochemical findings.

3. Model Systems for Specific Cells Two cell types, the macrophages and lymphocytes, have been studied in some detail and reliable sampling regimes have been developed for these and related cells. A selection of these models will now be presented. 3. I. Peritoneal macrophage

An experimental system designed for rat peritoneal macrophages was first proposed by Mayhew and Williams (1971) and subsequently exploited by Mayhew and Williams (1974a, b) Oakley and Mayhew (1978) and Mayhew (1979c). Mouse cells have been analysed using similar plans

246 . T. M. Mayhew and F. H. White

(Williams and Mayhew, 1973; Steinman et al., 1976; Woodward, 1978). The present model conforms to that used to analyse rat macrophages activated by exposure to Freund's adjuvants. General strategy. Peritoneal cells were harvested from groups of 12 animals by lavage, precipitated by centrifugation and fixed by immersion in glutaraldehyde with post-osmication. Morphometric analyses were based on systematic random samples of intact macrophages and of cells sectioned across the nucleus (nuclear-biased samples). Data were expressed in terms of the average cell in each population. Sampling procedures. The range of structural dimensions was such that sampling could be performed comfortably at only three levels of magnification. Stage I: Freshly withdrawn cells were spotted on to glass slides and not smeared. This was done to avoid the cell flattening which accompanies conventional smearing techniques. Cells were fixed in neutral formaldehyde and stained with haematoxylin and eosin or with toluidine blue. Preparations were scanned by light microscopy using a filar micrometer eyepiece. The magnification was 600 X. In this manner, estimates of maximum cell diameters were obtained. As a convenient approximation, mean cell volume was calculated for a sphere of mean cell diameter. Cell nuclei were anisodiametric and so irregular as to be unsuitable as a reference volume. Stage II: From a pool of pellet blocks, a sample of 3 blocks was selected at random (lottery method) and semithin araldite sections were cut. About 50 micrographs of toluidine blue-stained sections were recorded at a primary magnification of 500 X and printed at 2500 X. At this stage, estimates were made of the numbers of lysosomal granule profiles in nucleate macrophage profiles. From these counts and estimates of cytoplasmic profile areas obtained at stage III, areal densities of granule profiles were derived. These were to be compared with similar counts made at stage III. Stage Ill: Ultrathin sections were cut from the same blocks for electron microscopy. Some 30-40 micrographs were taken at 7500 X and 10000 X and printed at a final magnification of 28200 X. In general, sample sizes were considerably larger than the minima indicated by cumulative mean plots and/or calculation of relative standard errors (see Mayhew and Williams, 1974a). A coherent, quadratic test lattice of 1.0 em spacing was superposed on each nucleate cell profile. Volume densities of selected subcellular

Morphometry of Isolated Cells . 247 T able 1. Absolute stereological dat a for resident and adj uva nt -activated rat peritoneal macrophages (derived from data in May hew and W illiams, 1974a, b ; Mayh ew, 1979c) Co mpa rtment

Cell N ucleus Cytoplasm Mitochondria Granules Free ribosom es Rough ER Remaining cytoplasm

Surface ( ~lm2) Number Volume (urn") Resident Activa ted Resident Acti vated Resident Acti vat ed 126 32 95 8.1 60

144 36 108 9.1 9·5

4-4 76

4·5 85

Homogeneous granule diameter (nm): Surface amplification factor:

573 99

4 15 79 160 14° 1 676 9 61 4 60 000 474 000

Resident Resident

97·7 4·6

Activated Activated

126.2 3.2

features (T able I) were estimated by point counting. Volume densities of cytoplasmic organelles were referred to cytoplasm as the containing volume. On this morphometric model, " remaining cytoplasm" included free ribosomes, Golgi saccules and lamellae, smooth ER and the cytoplasmic ground substance. Counts were also made of the numbers of free ribosomes, lysosomes and mitochondria in order to determine organelle numerical densities. The sizes and size distributions of a homogeneous sub-population of granules (equated with primary lysosomes) were also reconstructed. A discontinuous line lattice (line length equi val ent to 1.08 urn) wa s employed to eva lua te cell and nucl ear surface/volume ratios. By mean s of these, cell surface amplification factors were compared.

Results and discussion. T able I illustrates the absolute stereological data obt ained for cells which normally reside in rat peritoneum and for cells activated by a single i.p. dose of Freund's complete adjuvant administered 5 da ys previously. Follo wing adjuvant activation there is a considerable drop in mean gra nule content and a nett depletion of plasmalemm a surface area. Since activated cells are large r th an normal, it follow s the y are also rounder. Increase in roundness appears to follow from the membrane interiorisation which accompanies endoc ytic activity .. The lower surface amplification factor and loss of surface membrane do not ad versely affect th e ability of

248 . T. M. Mayhew and F. H. White

activated cells to meet a subsequent phagocytic challenge: indeed, activated cells may be more efficient in this regard (Oakley and Mayhew, 197 8). Activated cells have fewer but generally larger granules. In part this reflects the formation of phagolysosomes, but adjuvant ingredients may also promote coalescence of primary lysosomes one with another. Activated cells have more mitochondria, rough ER and remaining cytoplasm, structural changes which reflect their altered metabolic status (Mayhew and Williams, 1974a, b). Differences between resident and activated cells are to be contrasted with those between cells in a given population. During maturation, cells grow and acquire substantially more plasmalemma so that larger cells are less rounded. Less mature cells also have fewer and smaller granules, fewer mitochondria and less rough ER. In fact, they more closely resemble their blood monocyte precursors (Mayhew, 1979c). Mouse peritoneal macrophages. Changes broadly similar to those seen in the rat accompany the in vivo activation of mouse cells by triolein (Williams and Mayhew, 1973). In an extension of this study, Woodward (1978) investigated morphometric differences between cells from germ-free and conventionally-reared mice. His results (Table 2) provide a similar picture of the quantitative morphology of resident cells in the conventional mouse. The average cell from germ-free animals is smaller with a less welldeveloped chondriome, less rough ER membrane and smaller granules. There is no change in surface amplification when average cells are compared with their equivalent spheres. The author suggests that macrophages from the germ-free mouse more faithfully represent the fully differentiated but non-activated cell. Steinman et al. (1976) have studied the rates at which pinosomes and surface membrane move into cultured macrophages during induced pino-

Table 2. Absolute stereological data for germ-free and conventionally-reared mouse peritoneal macrophages (derived from data in Woodward, 1978) Compartment

Cell Nucleus Cytoplasm Mitochondria Granules Rough ER

Volume (urn") Surface (urn") Number Germ-free Convention. Germ-free Convention. Germ-free Convention.

160 34 126 5.8 10·5

Surface amplification factor:

200 45

580 80

670

210

270

IIO

ISS 8·3 12·7

21',)0 Germ-free

4.2

1900

Conventional

4.2

Morphometry of Isolated Cells . 249

cytosis of horseradish peroxidase. Their analyses indicate that cells may interiorise the equivalent of their plasmalemma surface area every 33 minutes. It is postulated that at least some of the interiorised membrane components are subsequently recycled to the cell surface after shrinkage of nascent secondary lysosomes. 3.2. Alveolar macrophages

The first model system for alveolar macrophages was described for human cells (Jaubert et a1., 1974). More recently, Davies et al. (1977,1978) have provided a more detailed methodology for studying the effects of in vivo exposure of rats to tobacco smoke. The latter system is briefly described here. General strategy. Cells were obtained by bronchopulmonary lavage from groups of 30 animals. Suspensions of cells were fixed in glutaraldehyde/ OS04 and prepared as pellicles. Analyses were made using unbiased samples of cell profiles. Sampling procedures. From a reservoir of 30 pellicles, 9 were chosen at random. Each was cut into 6 blocks of which only 2-3 were sampled further. To meet experimental needs, four levels of magnification were adopted. Stage I: One field per semithin section was viewed under a light microscope having an automatic sampling stage and measurements were made of cell profile diameters. Actual cell diameters were reconstructed to obtain mean cell volumes (assuming cells were spherical). Stage II: One ultrathin section per grid was selected and all cell profiles on it were recorded. Electron micrographs of 136-155 cell profiles were printed at a final magnification of 12 400 X. These were evaluated using a square test lattice bearing 64 test points. Point and intersection counts were performed to obtain estimates of nucleocytoplasmic ratio and surface/volume ratios of cells and their nuclei. Stage Ill: A multipurpose coherent lattice (168 test points) was used to determine intracytoplasmic component densities at a magnification of 24000 X. Some 168-n8 cell profiles were measured to derive volume densities for mitochondria, lysosomes, lipid inclusions, myelin bodies and "remaining cytoplasm". The latter comprised all components not included in the other named compartments. Numerical densities of mitochondria were also calculated. Stage IV: Using the same lattice, some 302-324 profiles were analysed at 63400 X. Primary data was collected for surface density estimations: intracytoplasmic surface densities of mitochondrial outer

2S0 .

T. M. Mayhew and F. H. White

boundary and total inner membranes, smooth and rough ER and Golgi membranes.

Results and discussion. Typical results for control animals and for rats exposed to cigarette tobacco smoke for 90 consecutive days are given in Table 3. Smoke-exposed alveolar macrophages are considerably larger than normal. They have many more mitochondria and a correspondingly greater surface of mitochondrial membranes. They also have a greater volume of lysosomal material and a greater surface of organelle membranes involved in protein synthesis. Surface amplification is similar in both groups, the cell surface being roughly 70% greater than that of an equivalent sphere. As compared to macrophages from the peritoneal cavity, lung macrophages appear to be larger and to contain greater volumes of the main subcellular compartments. Though control cells seem to possess far fewer mitochondria than the average resident peritoneal macrophage, the mean volume of individual organelles is roughly five times bigger. The structural differences are consistent with the known biochemical and physiological differences between the two types of mononuclear phagocyte (e.g., Cohn, 1968). Human and Guinea Pig alveolar macrophages. Stereological definition of human cells has revealed two morphological classes, the "large and small mononuclear cells", which no doubt correspond to functional states of the

Table 3. Absolute stereological data for control and tobacco smoke-exposed rat alveolar macrophages (derived from data in Davies et al., 1978) Compartment

Cell Nucleus Cytoplasm Mitochondria Lysosomes Lipid bodies Myelin bodies Remaining cytoplasm RoughER Smooth ER Golgi zones Surface amplification factor:

Volume (urn") Control Exposed 623 12 7

49 6

u68 126

Surface (~lm2) Control Exposed

59 2 149

9 24

139

355

213

344 1471 4°7

Number Control Exposed

162

1042

2S

S3

52

98

5

204

15

15

399

672

768 178

Control

1.7

Exposed

1.7

Morphometry of Isolated Cells . 25 1

same cell type (Jaubert et al., 1974). Large cells appear to be more mature, with a rather low nucleocytoplasmic ratio and much phagocytosed material. In contrast, small cells have fewer pinocytic vesicles and dense bodies, a smaller chondriome and a higher nucleocytoplasmic ratio. In fact, like small peritoneal macrophages, small alveolar macrophages resemble monocytes, Recent studies in the Guinea pig (Soranzo et al., 1978) suggest that alveolar macrophages may also be distinguished on the basis of cytochemical peroxidase activity. Cytochemical differences may also be correlated with morphometric ones: peroxidase-positive cells also had a significantly greater volume density of rough ER. 3.3. Lymph node-derived lymphocytes

There have been many morphometric studies of lymphocytic cells in various lymphoid and myeloid organs (e.g., Heiniger et a1., 1967; Abe et a1., 1973; Matter, 1975; Le Bouteiller et al., 1976). The model described below was developed to assess morphological changes accompanying the in vivo blast transformation of small lymphocytes in mouse axillary lymph nodes. The process was induced by sensitisation with dinitrochlorobenzene (Al-Hamdani et al., 1979a and unpublished results). General strategy. Lymph nodes from pairs of mice were sliced into thin strips radiating from the hilum, fixed in glutaraldehyde and post-osmicated, Nuclear-biased samples of unstimulated small lymphocytes, immunoblasts and stimulated (daughter) lymphocytes were evaluated. Stimulated cells were identified with the aid of autoradiographic labelling with tritiated thymidine. Sampling procedures. Two nodes per pair of animals were sampled and each node furnished 40-50 tissue blocks of which 10-12 were chosen randomly. These were trimmed down to paracortical (thymus-dependent) areas. Sections were cut systematically at intervals of more than 10 urn (the diameter of the largest cells present). Two levels of magnification were sufficient. Stage I: Under a light microscope, nominal magnification 500 X, a single random micrograph was recorded from each semithin section. A total of II2 micrographs was taken to represent the paracortex of each node. All were printed at a final magnification of 2450 X. Nuclear profile diameter measurements formed the basis for estimating nuclear and cellular volumes. The nucleus was treated as a sphere. Stage II: On the same regime, II2 micrographs of ultrathin sections were taken at 4000 X and printed at 19650 X. Different species of

252 . T. M. Mayhew and F. H. White

quadratic test lattice (spacings

I

em,

I

in and

0.2

in) were employed

to estimate component volume densities of interest. Cell surface/

volume ratios were determined using a discontinuous lattice of line length equivalent to o. 52 urn, Minimal sample sizes were assessed by means of relative standard errors and/or cumulative mean plots. Results and discussion. Table 4 provides some of our results relating to unstimulated [ymphocytes, immunoblasts from mice 'sensitised 4 days previously and stimulated lymphocytes from animals sensitised 6 days previously. Unstimulated lymphocytes have a surface area some 9% greater, on average, than that of an equivalent sphere. The average immunoblast has almost six times the volume and over three times the surface area. This surface is also about 9% greater than that of a sphere with the same volume. In fact, the immunoblast undergoes a differential hypertrophy of all measured subcellular compartments. Our overall results support the view that immunoblasts are engaged in higher levels of protein synthetic activity. In contrast to unstimulated cells, stimulated lymphocytes are larger and have more plasmalemma. Indeed, they have a surface considerably greater (31%) than an equivalent sphere and this change accompanies an alteration in functional status (Al-Harndani et a1., 1979a). Lymphocytes of the lymphomyeloid complex. In a planimetric study of ultrastructural differences between thymic and lymph node small lymphoTable 4. Absolute stereological data for unstimulated lymphocytes, immunoblasts and stimulated lymphocytes in mouse axillary lymph node paracortex (derived from data in Al-Hamdani et al., 1979a, b and unpublished results) Compartment

Volume (urn") Surface (urn'') Unstimulated Immunob1. Stimu1. Unstimulated Immunob1. Stimu1.

Cell Nucleus Euchromatin Heterochromatin Nucleolus Cytoplasm Mitochondria Rough ER Golgi zones Remaining cytoplasm

68 4° 14·S 24·7 0·9 28 1.8

26

Surface amplification factor:

393 16S 102.2 S1.8 10·7 228 13.6 0·9 0·7 213

II4 63

89

283

ISO

SI 2·9

48

Unstimulated 1.1

Immunoblast 1.1

Stimulated 1.3

Morphometry of Isolated Cells . 253

cytes in mice, Heiniger et al. (1967) have shown that thymocytes have smaller nucleoli and a higher nucleocytoplasmic ratio. Small lymphocytes in the thymic cortex resemble those in bone marrow whereas the larger cells of the thymic medulla resemble small lymphocytes in the spleen (Abe et al., 1973). Moreover, the latter type seems to be morphologically, and probably functionally, more mature. Further attempts to distinguish immature from mature, immunocompetent thymocytes and to define thymic and bone marrow lymphocytes are described elsewhere (Matter, 1975; Le Bouteiller et al., 1976).

3·4. Peripheral blood-derived lymphocytes Although many authors have sought to characterise blood-derived lymphocytes and their progeny, the most comprehensive model system to date is that proffered by Petrzilka et al. (1978). This was originally designed to provide baseline stereological data on small, non-activated T-Iymphocytes in human peripheral blood. General strategy. Four adult males were used. Relatively pure (about 95%) populations of Tvlymphocytes separated on nylon wool columns were prefixed in glutaraldehyde/paraformaldehyde, sedimented by centrifugation and postfixed in osmic acid. Morphometric data were collected from unbiased samples. Sampling procedures. All measurements were made on electron micrographs. For each individual, 3 pellet blocks were chosen at random. One ultrathin section from each block was sampled at three levels. Stage I: A systematic random sample of 12 micrographs was taken from each section at 913 X and evaluated at a final magnification of I0440 X. Measurements of nuclear size and volume densities of nuclear components were made using a coherent double test lattice (IOO coarse and 2500 fine test points), Relative surface areas of cells and nuclei were also determined. Stage II: Micrographs within the same reference areas as stage I (i.e., 12 from each section) were recorded at 4760 X and analysed at 54430 X. A second double lattice (99 coarse: 891 fine test points) was employed to estimate cytoplasmic volume, surface and numerical densities. Stage III: Nine micrographs from each section were selected from areas sampled at stage II. These were recorded at 6120 X and printed at 70050 X. Ribosomal numerical densities were estimated with the aid of the second double lattice. 17

Path Res Pract. Vol. 166

254 . T. M. Mayhew and F. H. White Table 5. Absolute stereological data for human peripheral blood-derived small T-Iymphocytes (derived from data in Petrzilka et al., 1978) Compartment

Volume (urn")

Surface (flm2 )

104 48 15.6

128

Cell Nucleus Euchromatin Hererochromatin Nucleolus Cytoplasm Mitochondria Lysosomes Multivesicular bodies Dense bodies Lipid droplets Vesicles Filaments Rough ER Golgi zones Free ribosomes Ground substance Surface amplification factor:

Number

72

31.8

0·5 56 4. 6

0·3 0.1 0.01

0.05 0.06 0.03 0·7

30 4·3 2.8 0.6 0·3 63

0.2

231 100 50 1.2

Results and discussion. Baseline data provided in Table 5 partly confirm results obtained by others (Douglas et aI., 1973; Soren and Biberfeld, 1973). Unfortunately, they are not strictly comparable to those obtained for lymphocytes in mouse lymph node paracortices (Al-Hamdani et aI., 1979a). However, from similarly enriched human T-cell populations we have been able to show that blast transformation in vitro and in vivo is broadly similar in mice and men (Al-Hamdani et aI., 1979b).

4. Further Cell Models Each model described above was tailored to suit a specific type of cell and, taken together, the models serve to illustrate general principles of sampling and model design. In fact, many other types of isolated cell have been the objects of morphometric analysis, though not in such detail as above. Table 6 reviews some of these cell types.

5. Concluding Remarks The examples given above illustrate the practicality of applying stereological methods to isolated cells. Provided attention is given to various

Morphometry of Isol at ed C ells . 255 T ab le 6.

Mo rphom et ri c stud ies of o ther types of isolat ed cell Authors (D at e)

Objecti ves of th e in vest igation

C pUT ype

Sp ecies

M ast cells

Hela nder and Bloom Estima ti ng gr anule number and cont ent of histamine, heparin and (1974) y-hy d roxy tryp rami ne Consequ ences of inh ibit ion of Seglen and Re ith R at pr otein d egradat ion by am monia (197 6) (lysosoma l swelli ng) Fossum and Gautvik Effects of thyroliberin on proRat lact in synthesis and on vo lume and (1977) surf a ce of RER an d Gol gi Evalua ting th e ult ra-cyt ochemical Mouse W ollweber a nd stai ni ng of ca rb ohydrate sur face Gu inea pig Fr it sch (1975) receptors Membrane flow during pinocytosis Steinman et al. Mou se induced by hors eradish peroxidase (1976) Mi tocho ndrial growth an d d iv ision Man P osakon y et aI. d uring the cell cycle (1977) T et rah ymena K olb-Bachofen a nd Aspects of biogen esis o f mitochondria Vogell (1975) Tetrahymena Hofer et al. (1972) Correlating a ct iv ities of Krebs All gr omia Cycle en zyme s wi th mitochondrial Laby rinthula vol ume Trichom on as Nielsen and Di emer Repl ication of chroma ti c gra nules in va rious ph ases of cell gr owth (1976) Trypanosoma H ecker et al . (1972) Struc tura l definition of pleomorphic forms (slende r, intermedi ate and stu mpy) in rat blo od

H epatocyt es

Pitu icytes

P eritoneal cells, ascitic tumor

cells Fibroblasts (L cells) H eLa cells P rotozoa (cilia te) Protozoa (cilia te) P rotozoa (flage lla te) Protozoa (fla gellate)

Rat

sources of error, the methods will provide reliable and valid data. The main types of error are considered c1sewhere (Weib el, 1969 ; Elias et al., 197 I ; Mayhew and Cruz, 197 3, 1974). The correct design of sampling procedures should produce a regime which is optimal in terms of efficiency, representativeness and precision (W eibel, 1969, 1970; Shay, 1975). If the population is a mixture of cell types, it may be necessary to select cells sectioned through the nucleus in ord er to make recognition easier. This is especially true if there is no cytochemical identification. Stereology is of great value for objecti ve definition of isolated cells in normal, experimental and pathological states although few cell types have been analysed in detail. In fact , the isolated cell is a favourable model for the pathologist because representati ve sampling is relati vely easy. There is seldom anisotropy of structure such as is found in other model systems (e.g., skeletal muscle, kidney). Moreo ver, normal and pathological cells may be

256 . T . M. Mayhew and F. H . White

sampled using the same basic procedure. This is not the case in certain models where topographical features alter in the pathological condition (e.g., White et a1., 1980). A further advantage arises for isolated cells which can be prepared as comparatively pure populations. Both biochemical and structural data can then be related directly to the average cell of a particular type, thereby providing a more satisfactory basis for correlating structure and function than that achieved in conventional tissue preparations. Undoubtedly, our knowledge of aspects of the biology of normal and pathological isolated cells will improve as these and similar morphometric methods are applied to other models.

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Morphometry of Isolated Cells . 257 Dumont, A.: Ultrastructural study of the maturation of peritoneal macrophages in the hamster. J. Ultrastruct, Res. 29, 191-209 (1969) Elias, H., Hennig, A. and Schwartz, D. E.: Stereology: applications to biomedical research. Physiol. Rev. SI, IS8-200 (1971) Fossum, S. and Gautvik, K. M.: Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture. Stereology combined with non-parametrical statistics. Cell Tiss. Res. 184, 169-178 (1977) Fritsch, R. S.: Stereometrische Studie zur Oberflachendidire (s/v-Relationj von Zellen. Cytobiologie 3, 331-342 (1971) Hecker, H., Burri, P. H., Steiger, R. and Geigy, R.: Morphometric data on the ultrastructure of the pleomorphic bloodforms of Trypansoma brucei, Plimmer and Bradford, 1899. Acta trop. 29, 182-198 (1972) Heiniger, H. ]., Riedwyl, H., Giger, H., Sordar, B. and Cottier, H.: Ultrastructural differences between thymic and lymph node small lymphocytes of mice: nucleolar size and cytoplasmic volume. Blood 30, 288-300 (1967) Helander, H. F. and Bloom, G. D.: Quantitative analysis of mast cell structure. J. Microsc. lOO, 31S-321 (1974)

Hirsch, ]. G. and Fedorko, M. E.: Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" with uranyl acetate. ]. Cell BioI. 38, 61S-627 (1968) Hofer, H. W., Petre, D., Schwab-Stey, H. and Schwab, D.: Enzyme activity pattern and mitochondrio-cytoplasmic relations in protozoa. Comparative study of Tetrahymena pyriformis, Allgromia laticollaris and Labyrinthula coenocystis. J. Protozool. 19, S32-S4S (197 2)

James, V.: Stereological analyses of leukaemic cells. Brit. J. Haemat. 39, 17-24 (1978) Jaubert, F., Bignon, ]., Sebastien, P. and Butez, P.: Etude stereologique des cellules mononucleees alveolaires recueillies par lavage pulmonaire chez I'homme. Rev. franc, Mal. Respir. 2, 18-27 (1974) Jones, W. A. K. and Williams, M.. : Plasma cell maturation during the secondary immune response to a protein antigen. A combined stereological, cytochemical and autoradiographic study. In EMCON 72 Symposium Abstracts, yth Eur. Congr. EM, Manchester, p. II7 (1972) Kolb-Bachofen, V. and Vogell, W.: Mitochondrial proliferation in synchronized cells of Tetrahymena pyriformis. A morphometric study by electron microscopy on the biogenesis of mitochondria during the cell cycle. Expl. Cell Res. 94, 9S-IOS (197S) Konwinski, M. and Kozlowski, T.: Morphometric study of normal and phytohemagglutinin-stimulated lymphocytes. Z. Zellforsch. 129, S0D-S07 (1972) Le Bouteiller, Ph., Kinsky, R. G., Vujanovic, N., Due, H. T. and Voisin, G. A.: Morp'iological differences between thymus- and bone marrow-derived lymphocytes. II. An electron microscopic and experimental study in unstimulated mice. Differentiation 6, 12S-141 (1976)

Litovitz, T. L. and Lutzner, M. A.: Quantitative measurements of blood lymphocytes from patients with chronic lymphocytic leukemia and the Sezary syndrome. ]. nat. Cancer Inst. 53, 7S-77 (1974) Matter, A.: Morphological definition of thymocyte subpopulations, Cell Tiss. Res. 158, 3 19-332 (197S)

Mayhew, T. M.: Isolated peritoneal macrophages: component-biased sampling. In: Stereological Methods for Biomorphometry. Weibel, E. R., Academic Press (in press) (1979a) Mayhew, T. M.: Basic stereological relationships for quantitative microscopical anatomy a simple systematic approach. J. Anat, (in press) (1979b)

258 . T. M. May hew and F. H. Wh ite Mayh ew, T . M.: Quantitative ultrastructural features of maturing mononuclear phagocytes in rat peritoneal fluids. Experient ia 35, 390--392 (1979 c) Mayhew , T. M. and Cruz, L. M.: Stereo!ogical corre ction pr ocedures for estimating tru e volume proportions from biased samples. J. Microsc. 99, 287-299 (1973) Ma yh ew, T. M. and Cruz Orive, L. M.: Ca ve at on th e use of th e Del esse pr inciple of areal ana lysis for estimat ing component volume densities. J. Micro sc. I02 , 195-207 (1974) May hew, T. M. and W illiams, M. A.: A compari son of t wo samp ling procedures for stereological analysis of cell pell ets. J. Micr osc. 94, 195-204 (1971) Mayhew , T . M. and Wi llia ms, M. A.: A qu antitative morphological anal ysis of ma cro ph age stimulati on. I. A study of subcellula r comp artm ent s a nd of the cell surface. Z. Zellf orsch. 147, 567-588 (1974a) May hew, T. M. and Will iam s, M. A.: A quantitative morphol ogical analysi s of macrophage stimulation. II. Changes in gra nule number, size and size distributions. Cell Tiss. Res. ISO, 529-543 (1974b) N ielsen, M. H. and Diemer, N. H .: The size, density and relati ve area of chromatic granules ("hydrogenosomes" ) in Trichomonas vaginal is Donn e from cultures in logarithmic and stationary growth. Cell Tiss. Res. 167, 461-465 (1976) O akl ey, L. and Mayhew, T . M.: Membr ane interiorization by ph agocytosi ng macrop hagesan ultrastructural morp hometric approach. Expe rienr ia 34, 1338-1 339 (1978) Petrzilka, G. E., Graf - De Beer, M. an d Schroeder, H . E.: Stercological mod el system for fre e cells and base-line dat a fo r hum an p eripheral bloo d-d erived small T -l ymphocyte s. Cell Tiss. Res. 192, 121-1 42 ( 1978) Posak on y, J. W., En gland, J. M. and Attardi, G.: Mitoch ondrial grow th and di vision during the cell cycle in H eLa cells. J. Cell Bio!' 74, 468-491 (1977) Reith, A., Barnard, T . and Rohr , H . P.: Ste reolo gy of cellula r reac tio n patterns. Crit. Rev. Toxico!' 4, 219-269 (1976) Rohr, H . P ., Oberholzer, M., Barts ch, G. and Keller, M.: Morphometr y in experiment al pathology: met hod s, baseline dat a an d appl ications. Int. Rev. exp o Path. I S, 233-32 5 (1976) Schrek, R. : Ultrastructu re of blo od lymphocytes from chroni c lymphocyti c and lymphosarcoma cell leukemia. J. nat . Can cer Inst. 48, 51-64 (1972) Seglen, P . and Reith , A.: Amm on ia inhibition of protein degrad at ion in isolated rat hep atocyres. Quantitati ve ultrastructural alterations in the lysosomal system. Exp. Cell Res. 100, 276-280 (1976) Shay, J.: Econom y of effo rt in elect ro n microscope morphometr y. Amer. J Path. 81, 503 -5 12 (1975) Sor anzo, M. R., Koerten , H . K. an:l Daems, W. Th.: Peroxidat ic act ivity and morphometric analysis of alveolar macrophages in the guinea pig. J. reti culoendoth, Soc. 23, 343-359 (197 8) Sor en, L. and Biberfeld, P .: Qu antitative studies on RNA accumulation in human PHAstimula ted lymphocytes during blast transformation. Exp . Cell Res. 79, 359- 367 (1973) Steinman , R. M., Brodie , S. E. and Cohn, Z. A.: Membran e flow during p inocytosis. A ster eologic ana lysis. J. Cell Bio!' 68, 665-687 (1976) Vassart, G. M., Dumont, J. E. an d Cantra ine, F. R . L. : Interp retat ion of surface aspects of cell sections. J. Cell Bio!' 49, 210--212 (1971) Weibel, E. R.: Stereolo gical pr inciples for morphometry in electron microscop ic cyto logy Int. R ev. Cytol. 26, 235-302 (1969) Weibel, E. R. : An automatic sampling stag e micro scope for ster eology. J. Micros c. 9 1 , 1-1 8 (1970)

Morphometry of Isolated Cells . 259 White, F. H., Mayhew, T. M. and Gohari, K.: The application of morphometric methods to investigations of normal and pathological stratified squamous epithelium. Path.: Res. and Pract. 166, 323-346 (1980) Williams, M. A. and Mayhew, T. M.: Quantitative microscopical studies of the mouse peritoneal macrophage following stimulation in vivo. Z. Zellforsch. 140, 187-202 (1973) Wollweber, L. and Fritsch, S.: Methodological approaches to the study of carbohydrate surface receptors on macrophages and tumor cells, Neoplasma 22, 157-162 (1975) Woodward, B.: A stereological ultrastructural study of peritoneal macrophages from germ-free and conventionally-reared mice, Cell Tiss. Res. 192, 157-166 (1978) Zucker-Franklin, D., Davidson, M. and Thomas, L.: The interaction of mycoplasmas with mammalian cells. II. Monocytes and lymphocytes, ]. expo Med. 124, 533-543 (1966)

Received March 10, 1979 . Accepted April 23, 1979

Key words: Morphometry - Stereology - Free cells - Cultured cells Electron Microscopy Dr. Terry M. Mayhew, Department of Human Biology and Anatomy, The University, Sheffield S 10 2 TN, UK