J. Cell Set. 45, 59-71 (1980)Printed in Great Britain © Company of Biologists Limited 1080

EFFECTS OF COLCHICINE ON ENDOCYTOSIS

OF HORSERADISH PEROXIDASE BY RAT

PERITONEAL MACROPHAGES

ANDRZEJ PIASEK* AND JOHAN THYBERGfDepartment of Histology, Karolinska Institutet, Box 60 400, S-104 01Stockholm, Sweden

SUMMARYHorseradish peroxidase (HRP) was used as an exogenous marker to study the effects of

microtubule-disruptive drugs on endocytosis in cultures of thioglycollate-elicited rat peritonealmacrophages. Colchicine and vinblastine, but not lumicolchicine or cytochalasin B, reducedHRP uptake by about 30-40 %. However, as determined by stereological measurements, thesize of the HRP-containing compartment within the cells remained unaltered. In both controlcells and cells treated with colchicine or vinblastine the HRP-reactive vesicles were preferen-tially located close to the dictyosomes (stacks of cisternae) despite the fact that the Golgicomplex was disorganized in the treated cells. These results suggest that intact cytoplasmicmicrotubules are required to maintain a normal rate of fluid phase endocytosis in macrophages.On the other hand, it seems as if microtubules are not necessary for the translocarion of newlyformed endocytic vesicles/lysosomes to the dictyosomes, from which they probably aresupplied with digestive enzymes.

INTRODUCTION

In an accompanying paper (Thyberg, Piasek & Moskalewski, 1980) it was shownthat colchicine but not lumicolchicine causes a characteristic disorganization of theGolgi complex and lysosomal system in cultured rat peritoneal macrophages andepiphyseal chondrocytes, indicating a role for cytoplasmic microtubules in arrangingthese organelle systems within the cells. Although the total specific activities ofthiamine pyrophosphatase, a Golgi-bound enzyme, and acid phosphatase, a GERLand lysosomal enzyme, remained unchanged, the morphological effects producedby colchicine are probably accompanied by functional disturbances. It has previ-ously been shown that colchicine inhibits secretion of proteoglycans and collagen inchondrocytes (Jansen & Bornstein, 1974; Lohmander et al. 1976). Similarly, thecellular uptake of horseradish peroxidase (HRP) from the culture medium is reducedby colchicine (Piasek & Thyberg, 1979).

With few exceptions microtubule-disruptive drugs have been found to inhibitsecretion also in other cell types (for references see Piasek & Thyberg, 1979), includingrelease of lysosomal enzymes from phagocytes (Weissmann, Dukor & Zurier, 1971).

• Present address: Department of Histology and Embryology, Institute of Biostructure,Medical School, 02-004 Warsaw, Poland.

f To whom reprint requests should be addressed.

5-2

60 A. Piasek andj. Thyberg

Concerning the role of microtubules in endocytosis, available data are more incon-sistent (Allison, 1973; Korn, 1975; Silverstein, Steinman & Cohn, 1977). Here, wehave used biochemical and cytochemical techniques to study the effects of colchicineand other drugs on uptake of HRP into rat peritoneal macrophages, a cell type withhigh endocytotic activity. For comparative purposes and as a complement to ourprevious study (Piasek & Thyberg, 1979), rat epiphyseal chondrocytes were also used.Stereological measurements were made to trace possible changes in the size of theHRP-reactive compartment within the cells.

MATERIALS AND METHODS

Reagents. Ham's medium F-12 and foetal calf serum were obtained from Gibco Bio-Cult,Glasgow, Scotland, benzylpenicillin and streptomycin sulphate from Glaxo, Gfiteborg,Sweden, and Bacto thioglycollate broth N I H from Difco Labs., Detroit, Mich., U.S.A.HEPES, TES, collagenase type I, type II horseradish peroxidase (HRP), vinblastine sulphate,cytochalasin B, diaminobenzidine tetra-HCl (DAB), and o-dianisidine di-HCl were purchasedfrom Sigma Chemical Co., St Louis, Mo., U.S.A. Coomassie brilliant blue G-250 and col-chicine were products of Serva Feinbiochemica, Heidelberg, FRG. Lumicolchicine wasprepared by irradiation of colchicine solutions with long-wave ultraviolet light (Wilson &Friedkin, 1966). Bovine serum albumin came from Biomed., Cracow, Poland.

Isolation and culture of cells. Thioglycollate-elicited peritoneal macrophages and epiphysealchondrocytes were isolated from Sprague—Dawley rats and seeded out as described in thepreceding paper (Thyberg et al. 1980). Cells were allowed to attach to the dishes overnight.They were then rinsed and supplied with fresh normal medium or media containing colchicine,vinblastine, lumicolchicine, cytochalasin B or dimethylsulphoxide (DMSO, 10/il/ml ofmedium; solvent for cytochalasin B). All drugs were used at a final concentration of 1 x io" 5 M.After 2 h the medium was changed once more and the cells exposed for 2 h to 1 mg HRP/mlof medium, again with or without drugs. The cultures were thereafter washed 3 times with fullmedium and 3 times with non-supplemented medium, the cells harvested with a rubberpoliceman and washed another 3 times with non-supplemented medium. After the final rinsethe cell pellets were resuspended in 0-05% Triton X-100 and homogenized in glass homo-genizers. The homogenates were centrifuged at 4000 g for 15 min, the supernatants collectedand protein measured with the Coomassie brilliant blue-binding method (Bradford, 1976),using bovine serum albumin as standard. The samples were then diluted with 005 % TritonX-100 to equal protein concentrations and HRP activity determined according to Steinman &Cohn (1972) as described previously (Piasek & Thyberg, 1979).

Electron microscopy. The cells were fixed in situ for 1 h in 2 % glutaraldehyde (histochemicalgrade; Polaron, Watford, England) in 0 1 M sodium cacodylate buffer, pH 7-3. After overnightrinsing in 0 1 M cacodylate buffer with 0 2 M sucrose, cell-bound HRP was demonstratedaccording to Graham & Karnovsky (1966). The cells were incubated at 23 °C in 50 mg/100 mlDAB in 0-05 M Tris buffer, pH 7-6, first for 15 min without H , O , and subsequently for 60 minwith o-oi % H , O t . After being removed from the dishes with a rubber policeman, transferred

Figs. 1—3. Macrophages cultured in normal medium and exposed to HRP for 2 h.Fig. 1. Survey micrograph showing the accumulation of HRP-reactive vesicles in the

area around the dictyosomes (d) of the Golgi complex. Most of the vesicles are repletewith reaction product. No reactivity is present on the cell surface, TO, mitochondria;n, nucleus. No section-staining, x 23000.

Fig. 2. Higher magnification showing the close relationship between HRP-reactivevesicles and the dicytosomes (d) of the Golgi complex, n, nucleus. Section stainedwith lead citrate, x 47 000.

Effects of colchidne on endocytosis

T

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-•£••

• ''"WWH^

- • *

62 A. Piasek andj. Thyberg

to small plastic tubes and pelleted, the cells were postfixed in i % osmium tetroxide in o-i Mcacodylate buffer, pH 7-3, for 2 h, treated with 0-5 % uranyl acetate in Veronal-acetate buffer,pH 5-0, for 1 h (Farquhar & Palade, 1965), dehydrated in ethanol and embedded in Spurr'sresin (1969). Thin sections were cut on an LKB Ultrotome I, left unstained or stained withlead citrate (Reynolds, 1963), and examined in a Philips EM 300 electron microscope operatedat 80 kV. Calibration of the magnification was made with a carbon replica grating (LaddResearch Industries Inc., Burlington, Vt., U.S.A.).

Stereology. Stereological measurements were made on macrophages from 2 separate experi-ments with different batches of cells. The cultures were fixed and prepared as described above.Random sampling was ensured by photographing all cells in a single section from each specimen.The micrographs were examined at a final magnification of 15 coo times using a multipurposetest system consisting of an equilateral triangular network. The length of the test line on themicrographs (10 mm) corresponded to 0-7 fim in the cells. Volume and surface densities ofHRP-reactive vesicles were determined by point and intersection counting (Weibel, 1969).

Table 1. Drug effects on endocytosis of HRP by cultured macrophages

Endocytosis of HRP

Treatment

ControlColchicineLumicolchicineVinblastineCytochalasin BDMSO (10 ul/ml of medium)

ng/mg protein

330O2306338o210632073286

% of control

1 0 0

701 0 2

6497

1 0 0

Macrophages were allowed to attach to the dishes overnight. They were then pretreatedwith drugs for 2 h and thereafter exposed for 2 h to HRP (1 mg/ml of medium), again with orwithout drugs. All drugs were used at 1 x 10 ~J M. Data represent means of duplicate cultures.

RESULTS

Macrophages

For details regarding the differences in fine structure between control cells andcolchicine-treated cells the reader is referred to the accompanying paper (Thyberget al. 1980). A low endogenous peroxidase activity was present in the macrophages. Itamounted to about o-1 % of the activity ingested during 2-h exposure to HRP

Fig. 3. Reaction product for HRP in a complex system of interconnected channelsin the cytoplasm. The outer cell surface (right) is free of reactivity, n, nucleus. Sectionstained with lead citrate, x 30000.Fig. 4. Cell surface of cultured macrophage as demonstrated by labelling withconcanavalin A-HRP according to Bernhard & Avrameas (1971). Dense reactionproduct is present on the microvillous surface of the cell and in some plasma membraneinvaginations. When a-methyl-D-mannoside was added together with concanavalin Ain the first step of the labelling procedure, the cells remained unstained. This indicatesthat the surface staining was due to carbohydrate-containing components bindingconcanavalin A, and not to unspecific adsorption of HRP to the plasma membrane.The lack of cytoplasmic staining shows that the cell does not contain appreciableamounts of endogenous peroxidase activity. No section-staining, x 16000.

Effects of colchidne on endocytosis

A. Piasek andj. Thyberg

r.-*Gi*-----".=-L

> > v * ' . V

V

• "• ?

d'

Effects of colchicine on endocytosis

/ • •

Fig. 7. Part of a macrophage from a culture treated with vinblastine for 2 h and thenexposed to HRP in the presence of vinblastine for another 2 h. HRP-reactivevesicles located in the vicinity of 2 dicytosomes (d) of the Golgi complex are demon-strated. No section-staining, x 47 coo*

(1 mg/ml of medium) and hence was negligible in these experiments. Cytochemically,enzyme reaction product was noted in only a small proportion of the cells (cf. Fig. 4).

Maerophages kept in normal medium and exposed to HRP for 2 h showed numerousenzyme-reactive vesicles (Figs. 1, 2). A few of the vesicles demonstrated only aperipheral rim of reaction product, while most of them were completely filled withsuch product. According to Steinman, Brodie & Cohn (1976), the former structuresrepresent newly formed endocytotic vesicles and the latter secondary lysosomes.

Figs. 5, 6. Maerophages treated with colchicine for 2 h and then exposed to HRPin the presence of colchicine for another 2 h.

Fig. 5. Survey micrograph showing groups of HRP-reactive vesicles widely dis-tributed in the cytoplasm, x 13000.

Fig. 6. Further detail of HRP-reactive vesicles located close to a dictyosome (d) ofthe Golgi complex. Two presumptive endocytotic vesicles (ev), i.e. vesicles with onlya peripheral rim of reactivity, are seen. One of them (ev^ lies in direct contact witha presumptive secondary lysosome, i.e. a vesicle completely filled with reactionproduct. The other (evt) seems to be in the process of fusion with a vesicle of thislatter type. Sections stained with lead citrate, x 42000.

66 A. Piasek andj. Thyberg

Because of the scarcity of vesicles with only peripheral staining and the difficultiessometimes encountered in distinguishing them from vesicles with a uniform densereactivity, we will treat these structures collectively as HRP-reactive vesicles.

Table 2. Stereological analysis of HRP-reactive vesicles in macrophages treated withcolchicine or vinblastine

Measurement

1. Points/cell profile2. Points on cytoplasm/

cell profile3. Cell intersections/

cell points4. Number of vesicles/

cell points5. Vesicle/cell points6. Vesicle/cell

intersections

X

124

97'3

0-386

O-222OO88

O-39O

ControlA

S.D.

5I-S42-4

0150

0-1240-064

0228

n

4 i

41

41

41

4 i

4 i

TreatmentA

Colchicine

X

1 2 4

95-6

O'3S4

0-2120-078

0405

_^

S.D.

44'437-0

0-172

0-1190056

0-254

^n

U S

U S

" 5

U S

" 5

" 5

Vinblastine1

X

117

88-2

0-304

0-2060-067

0-474

A

S.D.

42-734-1

0-124

0-1040039

Q-355

n

5 i

51

51

5 i

51

5 i

Macrophages were allowed to attach to the dishes overnight. They were then pretreatedwith drugs (1 x IO~5M) for 2 h and thereafter exposed for 2 h to HRP (1 mg/ml medium),again with or without drugs. Lines 1 and 2 give the relative cell and cytoplasmic volumes;line 3 relates the surface area of the cells to their volume; line 4 gives the numerical density ofHRP-reactive vesicles per unit area of cell in section; and lines 5 and 6 give the volume fractionand surface area of HRP-reactive vesicles as related to the whole cell.

Most of the HRP-reactive vesicles were located within or around the juxtanuclearregion occupied by the Golgi complex (Fig. 1). Although a very close spatial relation-ship between these vesicles and the dictyosomes was observed, reaction product wasnever detected within Golgi cisternae (Fig. 2). In some cells, HRP-positive materialwas found not only in vesicles of the type mentioned above but also in narrow,interconnected channels (Fig. 3). Since no enzyme reaction product was present onthe free cell surface, these structures were probably not communicating openly withthe exterior of the cell. One possibility is that they represent a special type of endo-cytotic vesicle formed by deep cell surface invaginations pinched off into the cyto-plasm (cf. Steinman, Silver & Cohn, 1974). This interpretation was supported by theabsence of structures as complex as those described above after labelling the cellsurface with concanavalin A and HRP (Fig. 4) according to Bernhard & Avrameas

In cells treated with colchicine or vinblastine the uptake of HRP was reduced to60-70% of that in the controls (Table 1). This inhibitory effect was highly repro-ducible from one experiment to the other, although the level of HRP uptake variedslightly from one batch of cells to the other. Moreover, the distribution of HRP-reactive vesicles within the cells was changed, with groups of such vesicles distributed

Effects of colchicine on endocytosis 67

throughout the cytoplasm (Fig. 5). This change correlated with the deranged organiza-tion of the Golgi complex (Thyberg et al. 1980) with a close spatial relationshipexisting between HRP-containing structures and dictyosomes also in the drug-treatedcells (Figs. 6, 7). HRP-containing channels of the type described in control cells wereonly rarely observed after treatment with the microtubule-disruptive drugs. Lumi-colchicine, cytochalasin B and DMSO affected neither the uptake of HRP (Table i),nor the distribution of HRP-reactive vesicles within the cells.

Table 3. Drug effects on endocytosis of HRP by cultured chondrocytes

Endocytosis of HRP

Treatment ng/mg protein % of control

Control 150 icoColchicine 1 x 10-* M 101 67

Chondrocytes were allowed to attach to the dishes overnight. They were then pretreated withcolchicine for 2 h and thereafter exposed for 2 h to HRP (1 mg/ml of medium), again with orwithout colchicine. Data represent means of duplicate cultures.

Macrophages treated with colchicine or vinblastine and exposed to HRP were usedfor stereological measurements. The results of this analysis are summarized inTable 2. Cell and cytoplasmic volume and cell surface area were not distinctlyaltered by the drug treatments. Likewise, no clear effects were noted on numericaldensity, fractional volume or fractional surface area of HRP-reactive vesicles.

Chondrocytes

No endogenous peroxidase activity was detected in the chondrocytes, either bio-chemically or cytochemically. On a per mg protein basis the HRP uptake of thechondrocytes was only about 4-5 % of that of the macrophages. As in the latter cells,colchicine inhibited HRP uptake by about 30-40% (Table 3). Cytochemically, thenumber of HRP-reactive vesicles was low in the chondrocytes as compared to themacrophages. The differences in localization of these vesicles between control cellsand cells treated with colchicine were, however, the same in both cell types (cf.Piasek & Thyberg, 1979).

DISCUSSION

Thioglycollate-elicited rat peritoneal macrophages and epiphyseal chondrocytesgrown in vitro were found to take up HRP from the culture medium at highlydifferent rates, reflecting their specializations as endocytotic and secretory cells,respectively. The endocytotic activity of the macrophages was thus more than 20times that of the chondrocytes on a per mg cell protein basis and more than 10 timeshigher on a per cell basis. The mode of HRP uptake was not determined here butprevious studies (Steinman & Cohn, 1972; Steinman et al. 1974; Kaplan & Nielsen,

68 A. Piasek andj. Thyberg

1978; Piasek & Thyberg, 1979) as well as the lack of reactivity on the cell surfacesuggest that the enzyme was mainly, if not solely, interiorized by fluid endocytosis.

In spite of the large differences in endocytotic activity, colchicine was found to givea 30-40 % reduction of HRP uptake in both cell types, a degree of inhibition similarto that observed in embryonic chick chondrocytes (Piasek & Thyberg, 1979). Themore extended studies made on the macrophages here showed that this inhibitoryeffect was also obtained with vinblastine, another microtubule-disruptive drug, butnot with lumicolchicine, a photoisomer of colchicine not binding to tubulin (Wilson& Bryan, 1974). These findings indicate that intact cytoplasmic microtubules areneeded to retain a normal level of fluid endocytosis in peritoneal macrophages. Thelack of effect of cytochalasin B suggests in turn that microfilaments are not importantin this respect. Possible penetration problems and the complexity of action of thisdrug (Pollard & Weihing, 1974) make this result difficult to interpret, however.

Stereological measurements showed that numerical density of HRP-reactivevesicles per unit area of cell in section, as well as their volume fraction and surfacearea as related to the whole cell, were not distinctly affected by treatment of themacrophages with colchicine or vinblastine (Table 2). Most of the vesicles conformedto the definition of secondary lysosomes as given by Steinman et al. (1976), who foundthat the secondary lysosome compartment was saturated in 45-60 min after theaddition of HRP to the cultures and remained constant in size for at least 3 h. Inagreement therewith, we were unable to detect any unlabelled secondary lysosomesafter 2-h exposure to HRP. The above-mentioned quantitative findings thereforesuggest that neither number nor size of the secondary lysosomes were changed by themicrotubule-disruptive drugs. Since the concentration of HRP in the culture mediumwas the same in all groups, the inhibited uptake of HRP caused by these drugs wasprobably due to a decreased rate of formation of endocytotic vesicles. Alternatively,the endocytotic vesicles formed in colchicine- and vinblastine-treated cells weresmaller than those in the controls. This did not seem to be the case, however, althoughno strict quantitative evaluation could be done because of the relative scarcity ofendocytotic vesicles as compared with lysosomes, and the difficulties often en-countered in clearly distinguishing these structures from each other. In either case,the rate of interiorization of the plasma membrane must have been reduced.

On the other hand, the distribution of HRP-reactive vesicles in the cells waschanged by colchicine and vinblastine. This alteration corresponded to the re-distribution of lysosomes as observed in the accompanying paper (Thyberg et al,1980). HRP-reactive vesicles were thus found within and around the juxtanuclearGolgi area in control cells and close to the dispersed dictyosomes in cells treated withthese drugs. It therefore seems as if the newly formed endocytotic vesicles/lysosomeshave a microtubule-independent ability to orient themselves adjacent to the dictyo-somes/GERL, from which they probably are supplied with digestive enzymes.Although no chemical studies on the rate of degradation of HRP were done here,pictures indicating fusion between endocytotic vesicles and lysosomes were asfrequent in colchicine- and vinblastine-treated as in control cells (Fig. 6). This is inagreement with previous studies on macrophages (Pesanti & Axline, 1975 a, b) and

Effects of colchicine on endocytosis 69

chondrocytes (Piasek & Thyberg, 1979), suggesting that intact cytoplasmic micro-tubules are not required for fusion between endocytotic vesicles and lysosomes,although the pattern of movement of the former within cells may be disturbed (Freed& Lebowitz, 1970; Bhisey & Freed, 1971).

In cultured chondrocytes microtubule-disruptive drugs have been found to inhibitboth secretion of extracellular matrix components and uptake of HRP from themedium (for references see the Introduction). This led us to propose that there is amicrotubule-dependent coupling between exo- and endocytosis, which at least partlycontrols the balance in transport of membrane material to and from the plasmalemma(Piasek & Thyberg, 1979). The present finding of an inhibitory effect of colchicineand vinblastine on uptake of HRP by cultured macrophages and the fact that suchdrugs inhibit lysosomal enzyme secretion in phagocytotic cells (Weissmann et al.1971; Malawista, 1975) suggest that a similar coupling may also exist in these celltypes. Although it has been found that microtubule-disruptive drugs impair secretionin a wide variety of systems (for references see Allison, 1973; Piasek & Thyberg, 1979),it has also been reported that colchicine stimulates secretion of neutral proteases byexplants of synovial tissue (Harris & Krane, 1971, 1972) and macrophages (Gordon& Werb, 1976), of steroids by adrenal tumour cells in culture (Temple & Wolff, 1973),and of endogenous pyrogen by leukocytes (Bodel, 1976). This raises the possibilityof the existence of alternative secretory pathways with diverse relationships to themicrotubular cytoskeleton.

When comparing our data on the role of microtubules and microfilaments inendocytosis with others published in the literature, one is struck by the large variationin results. Colchicine has been found to inhibit ingestion of thyroglobulin from thefollicular lumen of the thyroid gland (Williams & Wolff, 1972), of sucrose by macro-phages (Pesanti & Axline, 1975 a), and of lysosomal enzymes and sulphated proteo-glycans by fibroblasts (von Figura, Kresse, Meinhard & Holtfrerich, 1978). Concerningphagocytosis of larger particles, the results vary depending on what type of particleis added to the cells (Malawista, 1975). In the case of cytochalasin B, it has beensuggested that this drug inhibits endocytosis of large particles (phagocytosis) and offluid into large vacuoles (macropinocytosis) but not of smaller molecules, e.g. solubleproteins, into minute vesicles (micropinocytosis), visible only in the electron micro-scope (Allison, 1973; Klaus, 1973). Cytochalasin has been found, however, to inhibituptake of lysosomal enzymes and sulphated proteoglycans by fibroblasts (von Figura& Kresse, 1974). In trying to summarize these different observations it would seemas if microtubules generally play a role in endocytosis, with the exception, perhaps, ofphagocytosis of some larger particles. With microfilaments it may be the reverse,i.e. that they are most important in phagocytosis and macropinocytosis and less, ornot at all, involved in micropinocytosis.

We thank Karin Blomgren for technical assistance. Financial support was obtained from theSwedish Medical Research Council (proj. no. 03355), the King Gustaf V 80th Birthday Fund,the Swedish Society of Medical Sciences, the M. Bergvall Foundation, and from the funds ofKarolinska Instituted

70 A. Piasek andj. Thyberg

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{Received 14 January 1980)