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Response of the resident macrophage to concanavalin A
Copyright Q 1980 by Academic Prcs‘ Inc. -211 rights of reproducticn in any form reserved 0014-4R27/80/[email protected]
Experimental
Cell Research
128 ~~~~~~ 439454
NSE OF THE CONCANAVAL Alterations of Surface A40
Department
of Chemistry,
Harvard University, Cambridge, MA 021.38, USA
SUMMARY We have characterized certain resident guinea-pig peritoneal macrophage surface alterations fohllowing treatment with concanavahn A (ConA) and succinyl-ConA (S-Co&A). Studies employing scanning electron microscopy (SEM) have demonstrated that ConA, a tetrameric lectin, decreases dramatically the number of macrophage surface folds and ruffles although S-ConA, a dimeric derivative. is apparently not active. However, incubation of S-ConA-treated cells with rabbit anti-ConA (anti-ConA) restored this effect. The decrease in surface fol s could not be observed in the presence of a-methyl-n-mannoside (LuMM), a hapten sugar of ConA. We have performed several receptor-labeling transmission electron microscopy (TEM) studies employing ferritin-conjugated ConA (FT-ConA) and cationized ferritin (CF). These experiments indicate that the ConA receptor-FT-ConA complexes form (1) clusters on the plasmalemma and (2) adsorptive pinocytic vesicles lined with the ferritin label. At the same time scale, we have observed a redistribution of macro&rage surface anionic sites followine treatment with ConA or S-ConA ohrs antiComA but not S-Con% alone. The redistribution of anionic sites is abolished in the presence of nMM. The sneciticitv of the CF label was checked bv me-incubating cells with notv-&sine (PLL) or . _ neuramimdase and by employing normal ferhtin. These studies provide evidence which support the concept of a directed movement of surface charges during adsorptive pinocytosis. We discuss the possible relevance of this concept with regard to regional alterations of pH at the ~~a§rn~~rn~ and the contribution of these anions to the trans-pinosomal p gradient through the Donnan potential
Since the ~~Q~ee~~g experiment of Tsan & , the non-random movements of ce components during the endoess have become well established. ve demonstrated that tKan§po~ functions for amino urine bases, and nucleosides are luded from endocytosed cell surface. This work in a number of laboratories [Z--5]. In contrast to the behavior of ost sites, certain surface structures such as ConA receptors [6,7], Richus conz~~4nis &tin receptors [6]> and Fc receptors [$] are preferentially included in the phago-
cytssed membrane. Certai appear to be removed fro fo~lQwing ~~agQcytQ~i§
I Present address: Stauffer Laboratory for Physical Chemistry, Stanford University, Stanford. CA 94305. USA.
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H. R. Petty
[ 121. In some cases, it has been demonstrated that these selective movements are abolished by colchicine, indicating that microtubules may be involved in the redistribution of surface elements ([ 121, for a review see [13]). Berlin [14] has suggested that an important step in the phagocytic process may involve a lateral phase separation of phagocyte surface lipids. In a preceding publication, we have employed the laser Doppler technique of electrophoretic light scattering to measure the effects of ConA adsorptive pinocytosis on macrophage surface charge density [ 151. We have suggested that the alterations in the electrophoretic mobility distribution were due to the non-random movements of negatively charged surface moieties into the pinocytic vesicle. In the present work transmission and scanning electron microscopy (TEM and SEM) have been employed to assess cell surface events taking place during adsorptive pinocytosis. This communication describes additional experimental findings which support the concept of directed movements of charged surface groups. This study and our previous work [15] provide suggestive evidence that negatively charged groups on the plasma membrane are internalized and therefore may contribute to the trans-pinosomal membrane pH gradient through the Donnan potential. Moreover, a local accumulation of surface charge would be expected to decrease the pH in the vicinity of a surface patch. The possible physiological relevance of these concepts is discussed. MATERIALS
AND
METHODS
Materials ConA and tained from Cationized (FT-ConA),
oc-methyl-o-mannoside ((uMM) were obSigma Chemical Co. (St Louis, MO). ferritin (CF), ferritin-conjugated ConA normal horse spleen ferritin, and poly-L-
Exp Cell Res 128 (1980)
lysine (PLL) were obtained from Miles Laboratory (Elkhart, IN). FT-ConA was also obtained from Cappel Laboratory (Cochranville, PA). Phosphatebuffered saline (PBS) and all tissue culture media were obtained from GIBCO (Grand Island, NY). Glutaraldehyde, collidine buffer, and 0~0, were obtained from Polysciences, Inc. (Warrington, PA). Succinyl-ConA (S-ConA) was prepared ad modum Gunther et al.
[161. Cell preparation Resident peritoneal cells were collected from normal non-sensitized Hartley guinea pigs (Charles River Breeding Labs). The animals were sacrificed by cardiac nuncture followed bv cervical dislocation. The cells -were collected in COOml of cold RPM1 1640 (Gibco, Grand Island, NY). After an initial washing, the red cells were lysed by osmotic shock if necessary. They were composed of roughly 75% macrophages and 25% eosinophils. Eosinophils were removed according to the method of Boyum [17]. Differential counts were made with Wright’s stain and the viable percentage (-95 %) of cells was determined by trypan blue exclusion.
Sample preparation
for SEM
Macrophages were fixed in suspension with 1% glutaraldehvde in PBS for 15 min on ice followed bv 1 h at room temperature. The cells were then washed in 0.1 M collidine buffer and oosttixed for 1 h in 1% 0~0, in collidine buffer at&room temperature. The cells were dehydrated in an increasing series of ethanol in water with three changes in absolute ethanol. The cells were placed in modified BEEM capsules with a Nuclepore filter (Pleasanton, CA) (pore size 1.0 pm) affixed to one end [lS, 191. The macrophages were critical-point dried in CO,. Cells were dispersed on a stub coated with double-sticky tape prior to sputter coating with gold-palladium. Specimens were examined in the AMR-1OOa scanning electron microscope. Micrographs were taken with Polaroid type 55 P/N film.
Cationized ferritin labeling macrophage surface
of
The micro-distribution of anionic sites (cationized ferritin receptors) was determined by the method of Danon et al. [20]. Briefly, a 10% cell suspension of macrophages was treated with 0.5 ml of a 1: 4 dilution of CF stock solution (11.5 mg protein/ml) in PBS. The suspension was gently agitated and was allowed to stand at room temperature for 30 min. The macrophages were washed with PBS and prepared for TEM.
Ferritin-labeled
ConA treatment
Macrophages in RPM1 1640 were warmed to 37°C for 15 min. The cells were then incubated with FT-ConA at loo-250 pg/ml (concentration of stock solution was
Fig. 1. (A) SEM of an untreated resident guinea-pig macrophage. These cells possess numerous surface folds and ridges. (B) SEM of a resident macrophage exposed to ConA at 100 pg/ml for 10 min at 37°C. The cells were pre-incubated at 37% for 15 min. When cells were treated in this fashion in the presence of (uMM, they were indistinguishable from the untreated cells. (C) Representative SEM of an S-ConA-treated macrophage.-Following a pre-incubation period of 15 min, the cells were exposed to S-ConA at 100 pg/ml for IO min at 37°C. The macrophages examined in this ex-
periment were most similar to untreated cells. (% this experiment macrophages were expose :d to S-C as described in (C). Thereafter, the ceils were wa: and incubated with anti-ConA (100 &ml ) for 40 ! xease in The cells have undergone a dramaficdeb number of surface folds, although a few nnicrovilli blebs can be discerned. If S-ConA is e> duded i morph# this treatment, no alterarion in surface can be observed. (A, B) ~7800; (C) x7 300; X6200.
442
H. R. Petty
determined by the method of Lowry et al. [21] with bovine serum albumin as standard) for various periods of time.
Ultrathin
sections
Cells which had been treated with CF, FT-ConA, or the control samples were fixed with 1% glutaraldehyde in PBS on ice for IS min followed by 1 h at room temperature. The macrophages were post-fixed with 1% 0~0, in 0.1 M collidine buffer (pH 7.0) at room temperature for 1 h. The cells were dehydrated in acetone and embedded in Suurr’s resin 1221. The embedded cells were thin-sectioned on a Reich& ultramicrotome equipped with a diamond knife. In several cases the sections were stained with uranyl acetate and Reynolds lead citrate [23]. The thin sections were examined in a Philips EM 300 electron microscope.
RESULTS SEM We present first a series of experiments which establish the effect of ConA and receptor cross-linkage on the surface morphology of the resident peritoneal macrophage. An SEM of an untreated macrophage is shown in fig. 1A. These cells possess many surface folds and ridges. The cells are spherical in shape since they have not adhered to a substrate. In fig. 1B we show a representative micrograph of a cell which has been treated with ConA at a concentration of 100 pg/ml for 10 min at 37°C. The number of surface folds has dramatically decreased. This is consistent with the view that substantial amounts of membrane are being internalized. We have not observed extracellular blebs with the phasecontrast microscope. No effect could be observed when macrophages were incubated with ConA (100 pg/ml) and aMM (100 mM) (data not shown). This indicates that the effect is due to the specific binding of ConA to certain cell surface saccharides. Resident peritoneal macrophages are heterogeneous in cell size. We have been unable to detect a substantial (>15 %) increase in cell size following ConA treatment with SEM (7 difExp Cell
Res 128 (1980)
ferent experiments; at least 40-50 cellscarefully observed in each). This is consistent with a with preliminary experiments Coulter counter. Since the ConA molecule is tetravalent, it is of considerable interest to establish whether the morphological responses are associated with the valence of the ConA molecule. In order to approach this question a dimeric derivative, S-ConA, has been employed. A macrophage treated with SConA at 100 pg/ml for 10 min at 37°C is shown in fig. 1 C. The dramatic effect seen with ConA was not observed with S-ConA, although a slight alteration may have taken place. Qualitatively, this cell population was more similar to the untreated rather than the ConA-treated macrophages. Similar results were found with S-ConA and ConA at 50 Fg/ml. In order to further document this process, we have incubated S-ConA-treated cells with anti-ConA at 100 pglml for 40 min at 37°C. The results of this experiment are shown in fig. 1D. A few surface microvilli are present, representing a significant reduction. A similar experiment employing normal rabbit serum had no effect (data not shown). These data indicate that crosslinking of ConA receptors is an important step leading to the disappearance of surface folds. ConA receptor labeling In order to establish the sequence of events during the macrophage-ConA interaction, we have employed a ferritin-conjugated derivative of ConA to follow the ConAreceptor complex. A typical survey electron micrograph of an FT-ConA-treated resident peritoneal macrophage is shown in fig. 2. The cells were pre-incubated at 37°C for 15 min in a 5 % CO, - 95 % air incubator. In the example shown, resident macro-
Fig. 2. Survey TEM of an FT.ConA-treated resident guinea-pig peritoneal macrophage. Following a preincubation at 37°C for 15 min, the macrophages were exposed to FT-ConA at a concentration of 2.50 pg/ml for 2 min at 37°C. The cells were rapidly washed, then fixed with glutaraldebyde. The prominent nucleus is
apparent. Sevemi vesicles containing the ferritin label can be found (UYTOWS) along with surface ciusters of the ferritin label (tkngles). This section has been counterstained with uranyl acetate and lead citrate. x34000.
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H. R. Petty
phages were exposed to FT-ConA at a concentration of 250 pug/ml for 2 min at 37°C followed immediately by centrifugation for 15 set in a Beckman microfuge. The cells were rapidly washed with cold PBS and resuspended in PBS containing 1% glutaraldehyde (~30 set). The molecular weight of ferritin is lo6 while that of ConA is 105; since the conjugate is nominally 1 : 1, the effective ConA concentration is roughly 25 pg/ml. As can be seen in the micrograph, there are (1) diffusely labeled areas on the plasmalemma; (2) patches of the label on the membrane; and (3) possibly internal vesicles containing the label. This thinsection was counterstained with lead citrate and uranyl acetate to enhance the ultrastructural features. The free ribosomes present in the cytosol should not be confused with the ferritin label. In this regard it should be noted that ferritin is not present in peritoneal macrophages under normal conditions [24]. For clarity, we show an enlarged version of this cell in fig. 3. Incubations for longer periods of time (15-60 min) resulted in the appearance of cytoplasmic vacuoles and a decrease in surface labeling. The specificity of this label was assessed by incubating FT-ConA and oMM with macrophages. The binding of the conjugate to the cell surface was not significant; in addition, pinocytic vesicles containing label could not be found (data not shown). Although it would have been of interest to examine the in situ distribution of ConA receptors, we shall not report these studies since brief glutaraldehyde fixation reduced significantly surface binding. Hence, the distribution of the ferritin label upon fixed cells would not correspond to that of the unfixed cells. A similar problem was encountered by other workers [25]. Studies employing fluorescence microscopy have shown that uniformly distributed ConA reExp Cd
RPS 128 (1980)
ceptors are rapidly clustered and pinocytized by resident macrophages [26-291. Cationized ferritin labeling Although a number of papers dealing with the CF labeling of the macrophage surface have appeared [30-321, proper control experiments on the specificity and nature of this label have heretofore not been performed. In prior experiments, it has been a tacit assumption that all cell membranes are labeled by CF in the same fashion; this is not necessarily a reasonable assumption. Therefore, we have performed a series of control experiments. In all CF experiments that we shall present, the macrophages were fixed briefly with 1% glutaraldehyde in PBS for 15 min before treatment with CF. This is necessary since CF is known to cause a redistribution and internalization of CF receptors on the surface of living guinea-pig resident macrophages [32]. In fig. 4, we present a CF labeling experiment of an untreated resident macrophage. The CF is distributed in a uniform fashion. In order to establish the nature of this labeling on the macrophage, we have performed several control experiments including the treatment of macrophages with PLL and neuraminidase prior to CF labeling and we have substituted normal horse spleen ferritin in place of its cationized derivative. These experiments are similar to several previous reports [20, 33, 341. The results of these studies are given in figs 5-7. Macrophages were pre-incubated with PLL at 250 pg/ml for 20 min at room temperature prior to the addition of CF. The CF labeling was essentially eliminated in all cells examined (fig. 5). In a few rare instances we could locate a small amount of label on portions of the plasmalemma. We suggest that the PLL becomes adsorbed to the cell surface, thereby de-
Macrophage
smrface response
to ConA
445
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H. R. Petty
creasing the apparent surface charge. The ination of CF labeling would indicate LL compete for similar surs, i.e., negatively charged groups. cept is further strengthened by exsediments in which macrophages were prebated with neuraminidase. Cells were with neuraminidase from Cl. pert 2 U/ml for 70 min at 37°C (where 1 U will liberate 1 km01 of N-acetyl neuraminic acid per min at pH 5.0). The macrowere then washed several times S followed by the CF protocol. The resm%ts, shown in fig. 6, demonstrate that treatment significantly reduces CF ing. Since neuraminidase removes any sialic acid residues from the cell surce, this indicates that the negatively c acid is required for some of ng. In addition, we have shown ationic nature of CF is necesry for ~~tera~t~on. Fig. 7 shows an experito that of fig. 4 except that 4. TEM of a CF-labeled normal resident guineaDig peritoneal macrophage. The CF receptors are &stributed in a uniform fashion. (The thin sections in rhe CF labeling experiments were not counterstained.) Identical res& &ere obtained when glycine (10 m&l) was incubated for 20 min with alutaraldehvde-fixed cells. Glycine might be expected-to block free aidehyde groups on the cell surface. x 5 1000. Kg. 5. Following pre-incubation with PLL (250 ,ug/ml) at room temperature for 20 min, the macrophages were exposed to CF in the usual fashion. The CF labeling has been dramatically decreased. A small amount of labeling can be noted. The PLL probably adsorbs to Ihe cell surface. thereby inhibiting CF binding. This suggests that negatively charged surface groups are required for the CF-macrophage interaction. x 50 000. Fig. 6. Normal macrophages were pre-treated with neuraminidase followed bv CF treatment. The removal of negatively charged si&c acid residues decreases substantiallv the number of CF receptors. This supports the cdncept that CF binds to negatively charged s&ace groups and that sialic acid accounts for at least a portion of the CF receptors. x.50000, Fig. 7. Macrophages were exposed to normal horse spleen ferritin instead of its cationized derivative. The protocol was identical to that employed in the CF experiments. No surface labeling can be found. This indicates that the cationic nature of CF is required for interaction. X41 000.
Fig.
times. Therefore, one receptors shouk! clu
redict that CF
448
H. R. Petty
uniform charge
negative disiributlon
surface
decreased
Uniform
Charge
+Anti
-Con
A
Clusters
“local”
pH
of Charge
4L.K
non-diffusible onions contribute to tronsmembrane pH gradient through Donnon potential decrease in internal pH
Fig. 12. A possible model of events taking place during the adsorptive pinocytosis of CozA by the resident macrophage. Adsorptive Pinocytic Vesicle
live micrograph from this study is shown in their relative S-C fig. 9. It is clear the effects of ConA are due to its interactions with certain carbohydrates on the cell surface. 37°C followed by the fixation and In analogy with the previous studies, we protocol described above for Con have examined the effects of S-ConA upon demonstrates that ConA valence istribution of CF receptors. Fig. 10 surface cross-linking ortant parashows a typical micrograph from this ex- meters with regard to §tr~~~t~~~ of periment. We have not observed a sig- CF receptors. nificant redistribution of CF receptors or This concept has been extende cubating S-ConA-treated ceils with antiConA at 100 pglml for 10 min. The results Fig. 8. Resident macrophages were treated with ConA of this study are given in fig. f “i~ A number at 100 pg/ml for 2 min followed by rapid centrifugation, glutaraldehyde fixation and CF labeling. A cluster of of CF clusters may be discerned on t rhe CF label can be seen on the cell surface. This in- membrane. e effects are sub dicates that CF receptors are redistributed during less than those fo’see ConA adsorptive pinocytosis. X 12 500. Fig. 9. This experiment is identical to that of fig. 9 exis may be due to diffe cept tbat (rMM at 100 mM was included. A uniform and kinetics of charge partidistribution of CF receptors is observed. The re- mechanism distribution of these receptors noted above is due to tioning or to the different procedures emthe specific binding of ConA to certain cell surface ployed. In any case, this is consistent wi:h saccharides. x 5 1000. Fig. 10. Resident macrophages were exposed to the effects of cross-linking on the disD-iS-ConA (100 pg/ml) for 2 min at 37°C and processed as bution of CF receptors. The resuhs are described above. There is no redistribution of CF receptors. This suggests that ConA valence is an imsummarized in fig. 12 1 portant parameter with regard to CF receptor distribution. x51 000. Fig. ii. This experiment is identical to that of fig. 10 except that tRe S-ConA treated cells were washed and resuspended in the presence of anti-ConA (100 kg/ml) for 10 tin at 37°C. Several clusters of the CF label can be found. X51 000.
Phagocyte surface pe the &and ConA lead to a wide variety of
450
H. R. Petty
biochemical and morphological alterations of the cell. ConA is known to bind to specific receptors on the macrophage plasmalemma [35, 361. Events that follow binding include endocytic stimulation [37], the formation of cytoplasmic vesicles and vacuoles [26-29, 37-391, an augmentation of adenylate cyclase stimulation [40, 411, and the induction of plasminogen activator [42]. In addition, ConA treatment is known to stimulate both oxidative metabolism [43], perhaps through trans-membrane cation fluxes [44], and superoxide ion generation [45, 461. An early event in this process appears to be an alteration of the transmembrane potential [47]. The studies we have presented indicate that a lateral movement of anionic sites is also an early event. Our previous studies [15], employing the laser Doppler technique of electrophoretic light suggested that negatively scattering, charged surface groups were preferentially internalized during adsorptive pinocytosis. The principal results of the present studies are: (1) a ConA valence-dependent decrease of surface folds; (2) a redistribution and pinocytosis of ferritin-conjugated ConA; and (3) a valencedependent redistribution of anionic membrane sites stimulated by ConA. SEM studies have shown a carbohydrate specific and ConAvalence dependent decrease of surface folds and microvilli; this indicates that the crosslinking of certain receptors is important in this process. Recently, it has been shown that ConA treatment decreases the number of surface ruffles in a CHO cell line [48] and inflammatory macrophages [49]. Other workers have noted localized decreases of surface folds following phagocytosis (for a review see [50]). We have observed a redistribution and pinocytosis of ConA receptors utilizing the ferritin-tagged lectin. This response is Exp CellRes
128 (1980)
abolished in the presence of the hapten sugar aMM. Clusters of the ferritin molecule were observed on the plasma membrane and presumably in internal vesicles. This process occurred on a rapid time scale of less than 5 min, which is in agreement with studies employing fluorescent derivatives [26]. Our work constitutes the first detailed study of this system on the fine structural level. Employing the technique of Danon et al. [20], we have shown that the CF receptors are laterally redistributed in the macrophage membrane following ConA treatment (see fig. 12). The CF labeling technique was shown to be specific for the negatively charged macrophage surface and the positively charged CF molecule. Hence, anionic sites are labeled on the plasmalemma with CF. Previous studies have suggested that each CF molecule labels several anionic sites [49]. The ConA-induced alterations were studied at low incubation times in order to observe the surface distribution of anionic sites at the same time scale as ConA patch formation and internalization. Unfortunately, it is impossible to observe the internalization of anionic sites on the living macrophage because CF, a highly charged macromolecule, will alone cause the crosslinking and adsorptive pinocytosis of negatively charged surface groups on the resident macrophage [32]. The negative surface charge clusters probably represent at least in part incipient ConA pinocytic areas. This is in agreement with the electrophoresis studies [ 1.51. It is reasonable to suggest that free aldehyde groups are present on the plasmalemma following glutaraldehyde fixation. These free groups may introduce artifacts if quantitative surface charge data are sought. However, if one seeks qualitative information regarding the general distribution of
CF receptors
on the plasmalemma, the e quite useful. We have amounts of non-specific al ferritin. Grinnell et al. [51] have noted that glutaraldehyde induces alterations of CF eptors on certain types of membranes. T e authors suggest that st likely explanation is that glutaral‘masked’ anionic sites on ne. Although this could have ~o~t~bution to our results, we not important since glutarale expected to affect the conex~er~me~ta~ samples in similar cently,
it has been noted that ConA duces a non-uniform eptors in rat hepatom [52]. It would be of interest to know if addisurface alterations were taking is system. The lateral redistribution of CF rece tors following CF treat11s has been noted by [32-34, 53, 541. Howev se experiments seem to iologicai interest. In addition, revious authors Rave applied ical concepts to the erimental results ~ The escribes several simple ovement of charge may have relevance
sible role ~fs~rfa~e
tively
charge in the
charged
surface
odynamic considerations of the electrical double layer, it is e concentration of cations near a negatively charged surface is greater
id ionic stren difference of a-0 etween the surfac
452
H. R. Petty
the activity of certain enzymes of the plasma membrane. Factors which may contribute to the decreased pH of endocytic vesicles include (1) proton pumps; (2) intra-lysosomal hydrolytic enzymes which generate protons; and (3) a Donnan potential generated by the accumulation of non-diffusible anions. In recent years a substantial amount of evidence has been accumulated which demonstrates that a Donnan potential at least partially accounts for the lysosomal pH gradient [6164]. The inner surface of the secondary lysosomal bilayer leaflet is very rich in sialic acid; this has been demonstrated with both histochemical and biochemical techniques [61, 621. The lysosomal membrane is unique among subcellular membranes in its high sialic acid content [62] and demonstrates a selective cation permeability [63651. The movement of cell surface charge into endocytic vesicles would be expected to contribute to the Donnan potential of the pinosome due to the addition of non-diffusable anions. The pH of the primary lysosome would be expected to decrease following fusion with the endocytic vesicle. The laser light scattering and electron microscopy studies have demonstrated a non-random movement of surface charges during adsorptive pinocytosis in the resident macrophage. These studies provide experimental evidence which supports the concept that charged surface groups contribute to the Donnan potential of the pinosome; furthermore, the endocytic vesicles are enriched in negatively charged moieties, indicating that the effect is greater than that obtainable by random inclusion. Previous workers have speculated that sialic acid groups of the lysosome may be contributed from the plasma membrane [62]. Moreover, Hughs [66] has put forth Exp Cell Res 128(1980)
the hypothesis that the sialic acid originating at the plasmalemma induces the pH drop of the lysosome upon fusion. Although the Donnan potential model probably plays a significant role, it is not yet clear whether the internalized charge in the ConA system could account for the entire pH drop seen in other experiments [67, 681. However, the various models of lysosomal pH control are not mutually exclusive. For example, the initial Donnan potential-induced pH drop might activate hydrolytic enzymes and/or H+ pumps which could complete the pH gradient. Such a mechanism would be consistent with the time-dependent pH changes which have been observed [67]. The detailed molecular mechanism of the surface charge partitioning during ConA adsorptive pinocytosis has not yet been completely defined. The charged groups could be directly or indirectly linked to the ConA receptors. For example, it is possible that ConA and CF are markers for the same membrane components. If this hypothesis is correct, then one would expect that charged groups are preferentially internalized during latex bead phagocytosis, since macrophage ConA receptors are selectively included during this process [6, 71. This possibility suggests that the concepts discussed above might be applicable to the phagocytic response. Alternatively, the ConA and CF receptors could be distinct entities which respond to the endocytic stimulus of ConA in a similar fashion. It would be of considerable interest to distinguish between these possibilities and to apply this approach to the study of phagocytosis, lymphokine responses [69], and mast cell activation [70]. The author is grateful to MS Nancy Basore for her expert experimental assistance. I should like to thank Dr K. R. Miller and MS G. Miller Brody for their many helpful comments. I thank Mr E. Seling for his as-
acrophage sistance with the scanning electron microscopy. I am indebted to Dr B. R. Ware for his encouragement. advice, and support during this study. This work was supported by Grant lRO1 GM 23788 from the NIH.
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to ConA
453
3J. Hardy, 3, Skuteisky. E, Globerson. A & Dannon. D, J reticuloendo SQC19 (1976) 291, 32. Skutelsky. E & Hardy, By Exp cell res 10%(1976) 337. 33. G W & Hein. C E, J cell biol72 (1977) 34. Moller, P C 6%Chang, J P, Exp celi reb 114 (‘r978) 39. 35. Alien. J M. Cook, G M W & Poole. A R, Exp cell res 68 (1973) 464. 36. Mahucci, L. Nature new biol 233 (1971) 241. 37. Edelson. P J & Cohn. Z A. 3 exp med 140 (1974) i364. 38. Goldman, R. FEBS iett 46 (1974) 233. 39. Go1dman.R &Rar. A. EXD ceil res % 11975) 393. 40. Grunspan-Swirsky. A & Pick. E, Ceil immunol 45 (1979) 415. 41. Gemsa. 0. Steggemann. L, Till. G & Resch. K. J immunol i 19 (1977) 524. 42. Vassallli. J D, Hamilton, .I & Reich, E. Cell : 1 (1977) 695. 43. Romeo, D, Zabucchi, G & Rossi, F. 4aiure new hi01 243 (1973) I 11. 44. Romeo. ‘3, Zabucchi. G. Mianr, N & Rasci. F, Nature 253 (1975) 542. 45, Goldstein, I M, Cerqueira. Liad. s &I Kapian. H 3, J clin inves 46. Goldstein, I M, s, Kaplan, H B & Weissmann, G. J ciin invest 56 (1975) i 155. 47. Korchak, B-8M 84 Weissmann. G. Proc nati acad sci US 75 (!978) 3818, 48. Noonan, K D & Ukena. T, J cell sci 34 (!978) 103. 49” Petty, H R. Ph.D. dissertation, Harvard rjni\;ersiry (1479). 50. Walters. M N 1 & Papadimitriov, J M. CRC critical rev toxicol 5 (1978) 377. 5i. ‘nnell, F, Anderson. G W & Hackenbrock C R, ochia biophys acta 426 (1976) 772, 52. oiler. P C & Chang, J P, Eur j cancer 15 :1979) 63. 53. Skuteisky, E & Dannon, D. J ceil brci 7’ i ,976) 232. 54. Marikovsky, Y, hodadad, J K &Weinstein. R S. Exp ceil res 116 (1978) 191. 55. Hartley. G S & Roe, J W. Tram Faraday sot 36 (1940) !Q!. 54. Seaman, G V F, The red biood cell (ed D M Sergenor) Press, New
57. 58. 59. 60. 61. 62. 63. 64.
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edn,
vol.
2.
p.
1ii5.
Academic
York (1975). Segal. A W 6%Peters. T J. C&n sci moi med 52 (iti7) 429. 3, Fed proc 37 (1978) 2759 NOSeWorthy, J, Korchak. H & Karrovsky. M L. J cell physic1 79 (1972) 91. Tsan, F & McIntyre, PA, 5 exp med iC3 (i976) 1308. Henning, R, Biochim biophys acta 401 (1975) 307. Henning, R, Plattner, H & Stoffefel.i;d. B&him biophys acta 330 ( !973) 61. Goldman, R & Rottenberg, H. FEBS Bert 33 (1973) 233. Goldman, R, Lysosomes in biology ant! patho:ony (ed J T Dingle & R T Dean) vol. 5, p. 309. Nor&Holland, Amsterdam (1976).
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65. Reijngoud, D J & Tager, J M, FEBS lett 54 (1975) 76. 66. Hughs, R C, Membrane glycoproteins, p. 94. Butterworths, London (1976). 67. Jenson, M S & Bainton, D F, J cell biol 56 (1973) 379. 68. Jacques, Y V &Bainton, D F, Lab invest 39 (1978) 179.
Exp CellRes
128 (19801
69. Petty, HR, Ware, B R, Remold, H G & Rocklin, R E, J immunol 124 (1980) 381. 70. Petty, H R, Ware, B R &Wasserman, S I, Biophys j 30 (1980) 41. Received October 17, 1979 Revised version received March 4, 1980 Accepted March 7, 1980
Experimental
Cell Research
128 ~~~~~~ 439454
NSE OF THE CONCANAVAL Alterations of Surface A40
Department
of Chemistry,
Harvard University, Cambridge, MA 021.38, USA
SUMMARY We have characterized certain resident guinea-pig peritoneal macrophage surface alterations fohllowing treatment with concanavahn A (ConA) and succinyl-ConA (S-Co&A). Studies employing scanning electron microscopy (SEM) have demonstrated that ConA, a tetrameric lectin, decreases dramatically the number of macrophage surface folds and ruffles although S-ConA, a dimeric derivative. is apparently not active. However, incubation of S-ConA-treated cells with rabbit anti-ConA (anti-ConA) restored this effect. The decrease in surface fol s could not be observed in the presence of a-methyl-n-mannoside (LuMM), a hapten sugar of ConA. We have performed several receptor-labeling transmission electron microscopy (TEM) studies employing ferritin-conjugated ConA (FT-ConA) and cationized ferritin (CF). These experiments indicate that the ConA receptor-FT-ConA complexes form (1) clusters on the plasmalemma and (2) adsorptive pinocytic vesicles lined with the ferritin label. At the same time scale, we have observed a redistribution of macro&rage surface anionic sites followine treatment with ConA or S-ConA ohrs antiComA but not S-Con% alone. The redistribution of anionic sites is abolished in the presence of nMM. The sneciticitv of the CF label was checked bv me-incubating cells with notv-&sine (PLL) or . _ neuramimdase and by employing normal ferhtin. These studies provide evidence which support the concept of a directed movement of surface charges during adsorptive pinocytosis. We discuss the possible relevance of this concept with regard to regional alterations of pH at the ~~a§rn~~rn~ and the contribution of these anions to the trans-pinosomal p gradient through the Donnan potential
Since the ~~Q~ee~~g experiment of Tsan & , the non-random movements of ce components during the endoess have become well established. ve demonstrated that tKan§po~ functions for amino urine bases, and nucleosides are luded from endocytosed cell surface. This work in a number of laboratories [Z--5]. In contrast to the behavior of ost sites, certain surface structures such as ConA receptors [6,7], Richus conz~~4nis &tin receptors [6]> and Fc receptors [$] are preferentially included in the phago-
cytssed membrane. Certai appear to be removed fro fo~lQwing ~~agQcytQ~i§
I Present address: Stauffer Laboratory for Physical Chemistry, Stanford University, Stanford. CA 94305. USA.
440
H. R. Petty
[ 121. In some cases, it has been demonstrated that these selective movements are abolished by colchicine, indicating that microtubules may be involved in the redistribution of surface elements ([ 121, for a review see [13]). Berlin [14] has suggested that an important step in the phagocytic process may involve a lateral phase separation of phagocyte surface lipids. In a preceding publication, we have employed the laser Doppler technique of electrophoretic light scattering to measure the effects of ConA adsorptive pinocytosis on macrophage surface charge density [ 151. We have suggested that the alterations in the electrophoretic mobility distribution were due to the non-random movements of negatively charged surface moieties into the pinocytic vesicle. In the present work transmission and scanning electron microscopy (TEM and SEM) have been employed to assess cell surface events taking place during adsorptive pinocytosis. This communication describes additional experimental findings which support the concept of directed movements of charged surface groups. This study and our previous work [15] provide suggestive evidence that negatively charged groups on the plasma membrane are internalized and therefore may contribute to the trans-pinosomal membrane pH gradient through the Donnan potential. Moreover, a local accumulation of surface charge would be expected to decrease the pH in the vicinity of a surface patch. The possible physiological relevance of these concepts is discussed. MATERIALS
AND
METHODS
Materials ConA and tained from Cationized (FT-ConA),
oc-methyl-o-mannoside ((uMM) were obSigma Chemical Co. (St Louis, MO). ferritin (CF), ferritin-conjugated ConA normal horse spleen ferritin, and poly-L-
Exp Cell Res 128 (1980)
lysine (PLL) were obtained from Miles Laboratory (Elkhart, IN). FT-ConA was also obtained from Cappel Laboratory (Cochranville, PA). Phosphatebuffered saline (PBS) and all tissue culture media were obtained from GIBCO (Grand Island, NY). Glutaraldehyde, collidine buffer, and 0~0, were obtained from Polysciences, Inc. (Warrington, PA). Succinyl-ConA (S-ConA) was prepared ad modum Gunther et al.
[161. Cell preparation Resident peritoneal cells were collected from normal non-sensitized Hartley guinea pigs (Charles River Breeding Labs). The animals were sacrificed by cardiac nuncture followed bv cervical dislocation. The cells -were collected in COOml of cold RPM1 1640 (Gibco, Grand Island, NY). After an initial washing, the red cells were lysed by osmotic shock if necessary. They were composed of roughly 75% macrophages and 25% eosinophils. Eosinophils were removed according to the method of Boyum [17]. Differential counts were made with Wright’s stain and the viable percentage (-95 %) of cells was determined by trypan blue exclusion.
Sample preparation
for SEM
Macrophages were fixed in suspension with 1% glutaraldehvde in PBS for 15 min on ice followed bv 1 h at room temperature. The cells were then washed in 0.1 M collidine buffer and oosttixed for 1 h in 1% 0~0, in collidine buffer at&room temperature. The cells were dehydrated in an increasing series of ethanol in water with three changes in absolute ethanol. The cells were placed in modified BEEM capsules with a Nuclepore filter (Pleasanton, CA) (pore size 1.0 pm) affixed to one end [lS, 191. The macrophages were critical-point dried in CO,. Cells were dispersed on a stub coated with double-sticky tape prior to sputter coating with gold-palladium. Specimens were examined in the AMR-1OOa scanning electron microscope. Micrographs were taken with Polaroid type 55 P/N film.
Cationized ferritin labeling macrophage surface
of
The micro-distribution of anionic sites (cationized ferritin receptors) was determined by the method of Danon et al. [20]. Briefly, a 10% cell suspension of macrophages was treated with 0.5 ml of a 1: 4 dilution of CF stock solution (11.5 mg protein/ml) in PBS. The suspension was gently agitated and was allowed to stand at room temperature for 30 min. The macrophages were washed with PBS and prepared for TEM.
Ferritin-labeled
ConA treatment
Macrophages in RPM1 1640 were warmed to 37°C for 15 min. The cells were then incubated with FT-ConA at loo-250 pg/ml (concentration of stock solution was
Fig. 1. (A) SEM of an untreated resident guinea-pig macrophage. These cells possess numerous surface folds and ridges. (B) SEM of a resident macrophage exposed to ConA at 100 pg/ml for 10 min at 37°C. The cells were pre-incubated at 37% for 15 min. When cells were treated in this fashion in the presence of (uMM, they were indistinguishable from the untreated cells. (C) Representative SEM of an S-ConA-treated macrophage.-Following a pre-incubation period of 15 min, the cells were exposed to S-ConA at 100 pg/ml for IO min at 37°C. The macrophages examined in this ex-
periment were most similar to untreated cells. (% this experiment macrophages were expose :d to S-C as described in (C). Thereafter, the ceils were wa: and incubated with anti-ConA (100 &ml ) for 40 ! xease in The cells have undergone a dramaficdeb number of surface folds, although a few nnicrovilli blebs can be discerned. If S-ConA is e> duded i morph# this treatment, no alterarion in surface can be observed. (A, B) ~7800; (C) x7 300; X6200.
442
H. R. Petty
determined by the method of Lowry et al. [21] with bovine serum albumin as standard) for various periods of time.
Ultrathin
sections
Cells which had been treated with CF, FT-ConA, or the control samples were fixed with 1% glutaraldehyde in PBS on ice for IS min followed by 1 h at room temperature. The macrophages were post-fixed with 1% 0~0, in 0.1 M collidine buffer (pH 7.0) at room temperature for 1 h. The cells were dehydrated in acetone and embedded in Suurr’s resin 1221. The embedded cells were thin-sectioned on a Reich& ultramicrotome equipped with a diamond knife. In several cases the sections were stained with uranyl acetate and Reynolds lead citrate [23]. The thin sections were examined in a Philips EM 300 electron microscope.
RESULTS SEM We present first a series of experiments which establish the effect of ConA and receptor cross-linkage on the surface morphology of the resident peritoneal macrophage. An SEM of an untreated macrophage is shown in fig. 1A. These cells possess many surface folds and ridges. The cells are spherical in shape since they have not adhered to a substrate. In fig. 1B we show a representative micrograph of a cell which has been treated with ConA at a concentration of 100 pg/ml for 10 min at 37°C. The number of surface folds has dramatically decreased. This is consistent with the view that substantial amounts of membrane are being internalized. We have not observed extracellular blebs with the phasecontrast microscope. No effect could be observed when macrophages were incubated with ConA (100 pg/ml) and aMM (100 mM) (data not shown). This indicates that the effect is due to the specific binding of ConA to certain cell surface saccharides. Resident peritoneal macrophages are heterogeneous in cell size. We have been unable to detect a substantial (>15 %) increase in cell size following ConA treatment with SEM (7 difExp Cell
Res 128 (1980)
ferent experiments; at least 40-50 cellscarefully observed in each). This is consistent with a with preliminary experiments Coulter counter. Since the ConA molecule is tetravalent, it is of considerable interest to establish whether the morphological responses are associated with the valence of the ConA molecule. In order to approach this question a dimeric derivative, S-ConA, has been employed. A macrophage treated with SConA at 100 pg/ml for 10 min at 37°C is shown in fig. 1 C. The dramatic effect seen with ConA was not observed with S-ConA, although a slight alteration may have taken place. Qualitatively, this cell population was more similar to the untreated rather than the ConA-treated macrophages. Similar results were found with S-ConA and ConA at 50 Fg/ml. In order to further document this process, we have incubated S-ConA-treated cells with anti-ConA at 100 pglml for 40 min at 37°C. The results of this experiment are shown in fig. 1D. A few surface microvilli are present, representing a significant reduction. A similar experiment employing normal rabbit serum had no effect (data not shown). These data indicate that crosslinking of ConA receptors is an important step leading to the disappearance of surface folds. ConA receptor labeling In order to establish the sequence of events during the macrophage-ConA interaction, we have employed a ferritin-conjugated derivative of ConA to follow the ConAreceptor complex. A typical survey electron micrograph of an FT-ConA-treated resident peritoneal macrophage is shown in fig. 2. The cells were pre-incubated at 37°C for 15 min in a 5 % CO, - 95 % air incubator. In the example shown, resident macro-
Fig. 2. Survey TEM of an FT.ConA-treated resident guinea-pig peritoneal macrophage. Following a preincubation at 37°C for 15 min, the macrophages were exposed to FT-ConA at a concentration of 2.50 pg/ml for 2 min at 37°C. The cells were rapidly washed, then fixed with glutaraldebyde. The prominent nucleus is
apparent. Sevemi vesicles containing the ferritin label can be found (UYTOWS) along with surface ciusters of the ferritin label (tkngles). This section has been counterstained with uranyl acetate and lead citrate. x34000.
444
H. R. Petty
phages were exposed to FT-ConA at a concentration of 250 pug/ml for 2 min at 37°C followed immediately by centrifugation for 15 set in a Beckman microfuge. The cells were rapidly washed with cold PBS and resuspended in PBS containing 1% glutaraldehyde (~30 set). The molecular weight of ferritin is lo6 while that of ConA is 105; since the conjugate is nominally 1 : 1, the effective ConA concentration is roughly 25 pg/ml. As can be seen in the micrograph, there are (1) diffusely labeled areas on the plasmalemma; (2) patches of the label on the membrane; and (3) possibly internal vesicles containing the label. This thinsection was counterstained with lead citrate and uranyl acetate to enhance the ultrastructural features. The free ribosomes present in the cytosol should not be confused with the ferritin label. In this regard it should be noted that ferritin is not present in peritoneal macrophages under normal conditions [24]. For clarity, we show an enlarged version of this cell in fig. 3. Incubations for longer periods of time (15-60 min) resulted in the appearance of cytoplasmic vacuoles and a decrease in surface labeling. The specificity of this label was assessed by incubating FT-ConA and oMM with macrophages. The binding of the conjugate to the cell surface was not significant; in addition, pinocytic vesicles containing label could not be found (data not shown). Although it would have been of interest to examine the in situ distribution of ConA receptors, we shall not report these studies since brief glutaraldehyde fixation reduced significantly surface binding. Hence, the distribution of the ferritin label upon fixed cells would not correspond to that of the unfixed cells. A similar problem was encountered by other workers [25]. Studies employing fluorescence microscopy have shown that uniformly distributed ConA reExp Cd
RPS 128 (1980)
ceptors are rapidly clustered and pinocytized by resident macrophages [26-291. Cationized ferritin labeling Although a number of papers dealing with the CF labeling of the macrophage surface have appeared [30-321, proper control experiments on the specificity and nature of this label have heretofore not been performed. In prior experiments, it has been a tacit assumption that all cell membranes are labeled by CF in the same fashion; this is not necessarily a reasonable assumption. Therefore, we have performed a series of control experiments. In all CF experiments that we shall present, the macrophages were fixed briefly with 1% glutaraldehyde in PBS for 15 min before treatment with CF. This is necessary since CF is known to cause a redistribution and internalization of CF receptors on the surface of living guinea-pig resident macrophages [32]. In fig. 4, we present a CF labeling experiment of an untreated resident macrophage. The CF is distributed in a uniform fashion. In order to establish the nature of this labeling on the macrophage, we have performed several control experiments including the treatment of macrophages with PLL and neuraminidase prior to CF labeling and we have substituted normal horse spleen ferritin in place of its cationized derivative. These experiments are similar to several previous reports [20, 33, 341. The results of these studies are given in figs 5-7. Macrophages were pre-incubated with PLL at 250 pg/ml for 20 min at room temperature prior to the addition of CF. The CF labeling was essentially eliminated in all cells examined (fig. 5). In a few rare instances we could locate a small amount of label on portions of the plasmalemma. We suggest that the PLL becomes adsorbed to the cell surface, thereby de-
Macrophage
smrface response
to ConA
445
446
H. R. Petty
creasing the apparent surface charge. The ination of CF labeling would indicate LL compete for similar surs, i.e., negatively charged groups. cept is further strengthened by exsediments in which macrophages were prebated with neuraminidase. Cells were with neuraminidase from Cl. pert 2 U/ml for 70 min at 37°C (where 1 U will liberate 1 km01 of N-acetyl neuraminic acid per min at pH 5.0). The macrowere then washed several times S followed by the CF protocol. The resm%ts, shown in fig. 6, demonstrate that treatment significantly reduces CF ing. Since neuraminidase removes any sialic acid residues from the cell surce, this indicates that the negatively c acid is required for some of ng. In addition, we have shown ationic nature of CF is necesry for ~~tera~t~on. Fig. 7 shows an experito that of fig. 4 except that 4. TEM of a CF-labeled normal resident guineaDig peritoneal macrophage. The CF receptors are &stributed in a uniform fashion. (The thin sections in rhe CF labeling experiments were not counterstained.) Identical res& &ere obtained when glycine (10 m&l) was incubated for 20 min with alutaraldehvde-fixed cells. Glycine might be expected-to block free aidehyde groups on the cell surface. x 5 1000. Kg. 5. Following pre-incubation with PLL (250 ,ug/ml) at room temperature for 20 min, the macrophages were exposed to CF in the usual fashion. The CF labeling has been dramatically decreased. A small amount of labeling can be noted. The PLL probably adsorbs to Ihe cell surface. thereby inhibiting CF binding. This suggests that negatively charged surface groups are required for the CF-macrophage interaction. x 50 000. Fig. 6. Normal macrophages were pre-treated with neuraminidase followed bv CF treatment. The removal of negatively charged si&c acid residues decreases substantiallv the number of CF receptors. This supports the cdncept that CF binds to negatively charged s&ace groups and that sialic acid accounts for at least a portion of the CF receptors. x.50000, Fig. 7. Macrophages were exposed to normal horse spleen ferritin instead of its cationized derivative. The protocol was identical to that employed in the CF experiments. No surface labeling can be found. This indicates that the cationic nature of CF is required for interaction. X41 000.
Fig.
times. Therefore, one receptors shouk! clu
redict that CF
448
H. R. Petty
uniform charge
negative disiributlon
surface
decreased
Uniform
Charge
+Anti
-Con
A
Clusters
“local”
pH
of Charge
4L.K
non-diffusible onions contribute to tronsmembrane pH gradient through Donnon potential decrease in internal pH
Fig. 12. A possible model of events taking place during the adsorptive pinocytosis of CozA by the resident macrophage. Adsorptive Pinocytic Vesicle
live micrograph from this study is shown in their relative S-C fig. 9. It is clear the effects of ConA are due to its interactions with certain carbohydrates on the cell surface. 37°C followed by the fixation and In analogy with the previous studies, we protocol described above for Con have examined the effects of S-ConA upon demonstrates that ConA valence istribution of CF receptors. Fig. 10 surface cross-linking ortant parashows a typical micrograph from this ex- meters with regard to §tr~~~t~~~ of periment. We have not observed a sig- CF receptors. nificant redistribution of CF receptors or This concept has been extende cubating S-ConA-treated ceils with antiConA at 100 pglml for 10 min. The results Fig. 8. Resident macrophages were treated with ConA of this study are given in fig. f “i~ A number at 100 pg/ml for 2 min followed by rapid centrifugation, glutaraldehyde fixation and CF labeling. A cluster of of CF clusters may be discerned on t rhe CF label can be seen on the cell surface. This in- membrane. e effects are sub dicates that CF receptors are redistributed during less than those fo’see ConA adsorptive pinocytosis. X 12 500. Fig. 9. This experiment is identical to that of fig. 9 exis may be due to diffe cept tbat (rMM at 100 mM was included. A uniform and kinetics of charge partidistribution of CF receptors is observed. The re- mechanism distribution of these receptors noted above is due to tioning or to the different procedures emthe specific binding of ConA to certain cell surface ployed. In any case, this is consistent wi:h saccharides. x 5 1000. Fig. 10. Resident macrophages were exposed to the effects of cross-linking on the disD-iS-ConA (100 pg/ml) for 2 min at 37°C and processed as bution of CF receptors. The resuhs are described above. There is no redistribution of CF receptors. This suggests that ConA valence is an imsummarized in fig. 12 1 portant parameter with regard to CF receptor distribution. x51 000. Fig. ii. This experiment is identical to that of fig. 10 except that tRe S-ConA treated cells were washed and resuspended in the presence of anti-ConA (100 kg/ml) for 10 tin at 37°C. Several clusters of the CF label can be found. X51 000.
Phagocyte surface pe the &and ConA lead to a wide variety of
450
H. R. Petty
biochemical and morphological alterations of the cell. ConA is known to bind to specific receptors on the macrophage plasmalemma [35, 361. Events that follow binding include endocytic stimulation [37], the formation of cytoplasmic vesicles and vacuoles [26-29, 37-391, an augmentation of adenylate cyclase stimulation [40, 411, and the induction of plasminogen activator [42]. In addition, ConA treatment is known to stimulate both oxidative metabolism [43], perhaps through trans-membrane cation fluxes [44], and superoxide ion generation [45, 461. An early event in this process appears to be an alteration of the transmembrane potential [47]. The studies we have presented indicate that a lateral movement of anionic sites is also an early event. Our previous studies [15], employing the laser Doppler technique of electrophoretic light suggested that negatively scattering, charged surface groups were preferentially internalized during adsorptive pinocytosis. The principal results of the present studies are: (1) a ConA valence-dependent decrease of surface folds; (2) a redistribution and pinocytosis of ferritin-conjugated ConA; and (3) a valencedependent redistribution of anionic membrane sites stimulated by ConA. SEM studies have shown a carbohydrate specific and ConAvalence dependent decrease of surface folds and microvilli; this indicates that the crosslinking of certain receptors is important in this process. Recently, it has been shown that ConA treatment decreases the number of surface ruffles in a CHO cell line [48] and inflammatory macrophages [49]. Other workers have noted localized decreases of surface folds following phagocytosis (for a review see [50]). We have observed a redistribution and pinocytosis of ConA receptors utilizing the ferritin-tagged lectin. This response is Exp CellRes
128 (1980)
abolished in the presence of the hapten sugar aMM. Clusters of the ferritin molecule were observed on the plasma membrane and presumably in internal vesicles. This process occurred on a rapid time scale of less than 5 min, which is in agreement with studies employing fluorescent derivatives [26]. Our work constitutes the first detailed study of this system on the fine structural level. Employing the technique of Danon et al. [20], we have shown that the CF receptors are laterally redistributed in the macrophage membrane following ConA treatment (see fig. 12). The CF labeling technique was shown to be specific for the negatively charged macrophage surface and the positively charged CF molecule. Hence, anionic sites are labeled on the plasmalemma with CF. Previous studies have suggested that each CF molecule labels several anionic sites [49]. The ConA-induced alterations were studied at low incubation times in order to observe the surface distribution of anionic sites at the same time scale as ConA patch formation and internalization. Unfortunately, it is impossible to observe the internalization of anionic sites on the living macrophage because CF, a highly charged macromolecule, will alone cause the crosslinking and adsorptive pinocytosis of negatively charged surface groups on the resident macrophage [32]. The negative surface charge clusters probably represent at least in part incipient ConA pinocytic areas. This is in agreement with the electrophoresis studies [ 1.51. It is reasonable to suggest that free aldehyde groups are present on the plasmalemma following glutaraldehyde fixation. These free groups may introduce artifacts if quantitative surface charge data are sought. However, if one seeks qualitative information regarding the general distribution of
CF receptors
on the plasmalemma, the e quite useful. We have amounts of non-specific al ferritin. Grinnell et al. [51] have noted that glutaraldehyde induces alterations of CF eptors on certain types of membranes. T e authors suggest that st likely explanation is that glutaral‘masked’ anionic sites on ne. Although this could have ~o~t~bution to our results, we not important since glutarale expected to affect the conex~er~me~ta~ samples in similar cently,
it has been noted that ConA duces a non-uniform eptors in rat hepatom [52]. It would be of interest to know if addisurface alterations were taking is system. The lateral redistribution of CF rece tors following CF treat11s has been noted by [32-34, 53, 541. Howev se experiments seem to iologicai interest. In addition, revious authors Rave applied ical concepts to the erimental results ~ The escribes several simple ovement of charge may have relevance
sible role ~fs~rfa~e
tively
charge in the
charged
surface
odynamic considerations of the electrical double layer, it is e concentration of cations near a negatively charged surface is greater
id ionic stren difference of a-0 etween the surfac
452
H. R. Petty
the activity of certain enzymes of the plasma membrane. Factors which may contribute to the decreased pH of endocytic vesicles include (1) proton pumps; (2) intra-lysosomal hydrolytic enzymes which generate protons; and (3) a Donnan potential generated by the accumulation of non-diffusible anions. In recent years a substantial amount of evidence has been accumulated which demonstrates that a Donnan potential at least partially accounts for the lysosomal pH gradient [6164]. The inner surface of the secondary lysosomal bilayer leaflet is very rich in sialic acid; this has been demonstrated with both histochemical and biochemical techniques [61, 621. The lysosomal membrane is unique among subcellular membranes in its high sialic acid content [62] and demonstrates a selective cation permeability [63651. The movement of cell surface charge into endocytic vesicles would be expected to contribute to the Donnan potential of the pinosome due to the addition of non-diffusable anions. The pH of the primary lysosome would be expected to decrease following fusion with the endocytic vesicle. The laser light scattering and electron microscopy studies have demonstrated a non-random movement of surface charges during adsorptive pinocytosis in the resident macrophage. These studies provide experimental evidence which supports the concept that charged surface groups contribute to the Donnan potential of the pinosome; furthermore, the endocytic vesicles are enriched in negatively charged moieties, indicating that the effect is greater than that obtainable by random inclusion. Previous workers have speculated that sialic acid groups of the lysosome may be contributed from the plasma membrane [62]. Moreover, Hughs [66] has put forth Exp Cell Res 128(1980)
the hypothesis that the sialic acid originating at the plasmalemma induces the pH drop of the lysosome upon fusion. Although the Donnan potential model probably plays a significant role, it is not yet clear whether the internalized charge in the ConA system could account for the entire pH drop seen in other experiments [67, 681. However, the various models of lysosomal pH control are not mutually exclusive. For example, the initial Donnan potential-induced pH drop might activate hydrolytic enzymes and/or H+ pumps which could complete the pH gradient. Such a mechanism would be consistent with the time-dependent pH changes which have been observed [67]. The detailed molecular mechanism of the surface charge partitioning during ConA adsorptive pinocytosis has not yet been completely defined. The charged groups could be directly or indirectly linked to the ConA receptors. For example, it is possible that ConA and CF are markers for the same membrane components. If this hypothesis is correct, then one would expect that charged groups are preferentially internalized during latex bead phagocytosis, since macrophage ConA receptors are selectively included during this process [6, 71. This possibility suggests that the concepts discussed above might be applicable to the phagocytic response. Alternatively, the ConA and CF receptors could be distinct entities which respond to the endocytic stimulus of ConA in a similar fashion. It would be of considerable interest to distinguish between these possibilities and to apply this approach to the study of phagocytosis, lymphokine responses [69], and mast cell activation [70]. The author is grateful to MS Nancy Basore for her expert experimental assistance. I should like to thank Dr K. R. Miller and MS G. Miller Brody for their many helpful comments. I thank Mr E. Seling for his as-
acrophage sistance with the scanning electron microscopy. I am indebted to Dr B. R. Ware for his encouragement. advice, and support during this study. This work was supported by Grant lRO1 GM 23788 from the NIH.
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