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Differential effects of lectins mediating erythrocyte attachment and ingestion by macrophages
E.~perimerztal Cell Research 103 (1976) 279-294
DIFFERENTIAL
EFFECTS
ERYTHROCYTE
OF LECTINS
ATTACHMENT
AND
MEDIATING INGESTION
BY MACROPHAGES RACHEL GOLDMAN Department
of Membrane
Research,
and I. BURSUKER
Weiynann
Institlrre
of Science, Rehovor, Israel
SUMMARY Lectin-mediated interaction of erythrocytes and macrophages was brought about in two steps. Step I involved macrophage treatment with lectin. and step II is the incubation of lectin-treated macrophages with mouse erythrocytes. The extent and nature of lectin-mediated macrophage erythrocyte interaction was studied using concanavalin A (ConA), wheat germ (WGA), soybean (SBA) and waxbean (WBA) agglutinins. The parameters affecting the interaction were studied in detail with the first two lectins. Under comparable conditions of lectin interaction with macrophages (step I). WGA mediates rosette formation involving interaction with several times the number of erythrocytes than those interacting with ConA-treated macrophages. The interaction mediated by WGA reaches, at 37°C. a saturation value after 30 min of step II. whereas that mediated by ConA is still linear and exhibits half the amount of attached erythrocytes at 60 min. Cot&mediated attachment of erythrocytes is highly temperature-dependent being at 37°C twice that observed at 24°C. The temperature dependence of attachment is not affected by changes of either ConA concentration (5-40 pg/ml) or the temperature in step I. An optimum is observed, however. when the temperature of incubation in step I ranges between 14-18°C. WGA-mediated attachment of erythrocytes is markedly less temperaturesensitive, exhibiting 70% of optimal attachment already at 8°C. Only when the attachment phase foliows incubation with a low concentration of WGA (2 pg/ml) high temperature sensitivity is exhibited. At 3?“C, however, the number of attached erythrocytes is the same for macrophages treated with WGA at concentrations of 2. 5, 10 and 40 pg/rnI. ConA-mediated erythrocyte-macrophage interaction does not lead to erythrophagocytosis. When mediated by WGA, the attachment step is followed by a temperature-dependent ingestion step. i.e. 10% and 50% of the erythrocytes that attach to macrophages during the 60 min incubation at 24°C and 37°C. respectively. are ingested. There is a lag period of 10-20 min between attachment and ingestion implicating involvement of additional cellular processes preceding engulfment. Electron microscope images of areas of interactron of attached erythrocytes with macrophages indicate a significantly tighter binding (a thinner gap at membrane-membrane apposition areas) in the case of WGA-mediated rosette formation as compared with that established in ConA-mediated rosettes. Attachment via WGA is followed by a rapid change in the re!ative position of the attached erythrocytes on the macrophage, from a primary attachment at the distal peripheral regions of the cell, to a perinuclear position. In contrast, erythrocytes attached via ConA remain at the primary attachment point (at 37°C) for extended periods. This differential behaviour does not stem from effects of ConA on macrophages, since when yeast cells were attached to ConA treated macrophages. the yeast cells showed the same movement as that exhibited by erythrocyte when attached via WGA. The different interaction patterns of erythrocytes with macrophages coated with ConA and WGA can be fit:cd into the following working hypothesis: the number of WGA-binding sites on the plasma membrane of macrophages is at least three times that of ConA-binding sites. Stable cell-cell interactions involve multibridge formation at the contact area of the two cells and this involves a delicate balance between number of iectin-receptor conjugates and their aggregation state within the membrane phase. A certain amount of clustering is a prerequ;site for attachment, while a high degree of clustering reduces the chance of fruitful interactions. The engulfment step depends on the ability of membrane areas adjacent to primary contact area to establish additional stable bridges in the entire circumference of the attached cell. ConA-receptor conjugates appear to be less abundant and more aggregated within the membrane plane. preventing the completion of fruitful circumferential interaction of the adjacent membrane. WGA-receptor conjugates, being more abundant and apparently less aggregated are available at membrane areas needed for cell enclosure and provide the additional bridging without which engulfment does not take place. Change in relative position of attached erythrocytes seems to be a step in the manifold events occurring from attachment to ingestion.
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Goldman and Bursuker
The means by which phagocytes recognize and ingest have been a long-term pursuit in diverse fields. The mechanism for recognition and attachment is generally divided into two main categories, immune and nonimmune recognition. In spite of the extensive studies, the process still poses many riddles to the investigator. One of the most exciting questions is the way in which contact could trigger the events leading to formation of phagosomes and the molecular organizational events on the membrane level that precede it. A number of studies have established that though attachment of particles is a prerequisite for their ingestion by macrophages, attachment per se is not a sufficient signal for particle interiorization. In immune phagocytosis it was established that the attachment process is distinct from the ingestion process in both temperature dependence [l] and the degree of required opsonization, be it with IgG alone or IgG+ the C3 component of complement [ 11.It has been shown that the process is specific and limited to the segments of plasma membrane to which the ingested particles are bound [2] and that ingestion requires the sequential, circumferential interaction of particle bound ligands (IgG, IgM+CJ with specific plasma membrane receptors not involved in the initial attachment process [3]. Previous work in our laboratory has characterized a concanavalin A (ConA)mediated attachment and ingestion of fresh homologous erythrocytes and yeast cells with mouse peritoneal macrophages [4, 51. Erythrocyte- or yeast cell-macrophage rosettes were formed under conditions in which either the erythrocytes or yeast cells were ConA coated or the macrophages were ConA coated. The attachment of ConA-treated erythrocytes to macrophages was more efficient than the attachment of Exp Cell Res 103 f 1976)
erythrocytes to ConA-treated macrophages (M-ConA). In neither case, however, was the attachment followed by erythrocyte ingestion. Addition of ConA to preformed rosettes and further incubation at 37°C resulted in 100% ingestion of the attached erythrocytes [4]. It was argued that the additional ConA was needed for saturation of ConA-binding sites on the erythrocytes, a saturation that could not be achieved in erythrocyte suspensions. Yeast cells have a higher surface density of ConA-binding sites than erythrocytes [S]. Yeast cell ingestion was triggered in both, rosettes formed from interacting yeast cells coated with ConA with macrophages and in rosettes formed from yeast cells interacting with M-ConA. The extent and temperature dependence of ingestion under the two rosetting schemes, however, showed striking differences [5]. On the basis of the detailed studies concerning ConA-mediated yeast cell-macrophage interaction it was suggested that ingestion involves the recruitment of more ConA bridges at areas of membrane-yeast cell interaction than needed for a stable yeast cell attachment. It was also suggested that the mobility of ConA receptors in the plane of the plasma membrane whether free or ConA-conjugated was an important factor in triggering ingestion, overriding metabolic energy requirements as well as the activity of the cytoskeletal elements (microfilaments and microtubules). Lectin-binding sites in eukaryotic cells represent a heterogeneous population of glycoproteins [6]. Previous work from our laboratory has characterized the differential response of macrophages to various lectins [7]. The agglutination patterns of erythrocytes, and fibroblasts in response to lectin binding has indicated that different lectins
Differential lectin effects in erythrophagocytosis MATERIALS
,.,,i /‘/i
281
AND METHODS
Media Dulbecco’s modified Eagle’s meditim (medium) was supplemented with 100 U/ml penicillin and 100 @g/ml streptomycin, and sterilized by Millipore fiitration. Medium, heat-inactivated new-born calf serum (serum) and (Dulbecco’s) phosphate-buffered saline (!PBSj were obtained from Grand Island Biological Co. ~N.Y.
t //J 500 ?
Lectins and their inhibitors )
/ 2
3
4
5
cont. of erythrocytes suspensions incubated with macrophages, x10-‘; ordinate: no. of erythrocytes attached per 100 macrophages. Lectin-mediated ervthrocvte-macronhage rosette formation. Macrophages were coated wiih either WGA (O-O) or ConA (0-O) (20 fig/ml of lectin. 30 min. 14’C). Subsequent to ~2 ‘wash-in PBS lectin-treated macrophages were incubated with erythrocytes (24”C, 60 min).
Fig. 1. Abscissa:
generate associations differing in their strength [S], concentration and temperature dependency [9, lo]. It was also established that the number of lectin binding sites/cell and the association constants are markedly different [lo]. The experiments reported in this paper were designed to assess the interaction pattern of erythrocytes with lectin-treated macrophages and to find out whether lectinmediated interactions would be able to trigger erythrophagocytosis. The lectins used in the study were ConA, wheat germ (WGN, soybean (SBA) and waxbean (WBA) agglutinins. The analysis concentrated in particular on the contribution of parameters such as temperature, lectin concentration, erythrocyte concentration, and duration of interaction to the formation of erythrocyte-macrophage rosettes, to subsequent erythrocyte ingestion (WGAmediated) and to the spatial arrangement of the erythrocytes relative to the cell periphery and nucleus.
ConA, twice crystallized, was obtained from MilesYeda (Rehovot, Israel) and WGA. WBA and SBA. prepared by affinity chromatography [7], were a gift of Dr R. Lotan. a-Methyl-D-mannopyranoside ((YMM) and N-acetyl-o-glucosamine were obtained from Pfanstiehl Labs. Inc., Waukegan.
Collection and cultivation qf macrophages Peritoneal macrophages were aseptically collected from BALE/c strain male mice, weighing 20-25 g. Peritoneal exudate cells suspended in medium were allowed to attach (5x 10” cells in 0.15 ml. 60 min, 37’9 on either 25 mm diameter Coming cover glasses or cover glasses coated with a thin sheet of parlodion [ll]. The cover glasses were placed in 35 x ld mm Faicon tissue culture dishes (Falcon Plastics Div. Bioquest. Oxnard, Calif.). After the phase of cell attachment the plates were thoroughly rinsed in PBS IO remove non-adhering cells. Macrophages were then cul-tivated for 48 h at 37°C in 2 ml of 20% serum ir. Dulbecco’s modified Eagle’s medium, in a CO,incubator (5 ‘% COTair mixture). Culture medium was changed once after 24 h incubation.
Macrophage interaction uqith iectins Macrophage monolayers were twice washed in PBS and1incubated with the specified lectin at the concentrations, temperature and time specified in the legends to the corresponding figures, and subsequentiy washed twice in PBS.
Erythrocyte-macrophage interaction Freshly obtained mouse blood was anticoagulated iyith citrate phosphate dextrose solution (Travenol Lab. Ltd, Ashdod, Israel), then washed with PBS containing 5 mM of glucose (PBS-glucose) to remove plabma and buffy coat. Unless otherwise specified. 1 ml of an erythrocyte suspension (10’ cells/ml PBS-glucasc) was added to lectin-treated macrophage monoiayers. At the end of the incubation period non-adherent erythrocytes were removed by twice-washing with PBS on a rotatory shaker (100 rotations/min, Clinical Rotator, Eberbach Corp. Ann Arbor. Mich.i. For evaluation of erythrocyte attachment and ingestion, cells were fixed (2% glutaraldehyde in PBS, 30 min at 4°C) and stained (May-GrtiwaldGiemsa).
282
Goldman and Bursuker RESULTS
time (mm); ordinate: (left) no. erythrocytes interacting with 100 macrophages (0-O); (rigkt) % of ingested erythrocytes out of total macrophage associated erythrocytes (O-O). Time dependence of lectin-mediated erythrocytemacrophage interaction. Macrophages were coated (30 min, 14°C. 20 pg/ml) with either (+4) ConA or (B) WGA. O-Cl, WGA-mediated rosettes were formed at 14°C (60 min); washed twice in PBS and transferred to fresh PBS+glucose at 37°C for the specified times; O-0, % ingestion.
Fig. 2. Abscissa:
Erythrocytes attached and ingested per 100 macrophages were enumerated in triplicate cultures. Attached erythrocytes were readily distinguished from ingested erythrocytes by a colour change, glassy appearance and irregular contour of the latter. For electron microscopy cells cultivated on parlodion sheets were allowed to interact with lectins and erythrocytes as specified in the figures. The interacting cells were then processed as described [ 1l] and embedded in epoxy resin according to Spurr [12]. Thin sectioning was obtained with Sorvall, Porter-Blum MT 2-B ultramicrotome and the sections were analysed in a Philips 300 electron microscope operated at 80 kV. The sections were stained prior to visualization with lead citrate.
Treatment of macrophages and erythrocytes with neuraminidase
loot
Neuraminidase from Vibrio comnta was obtained from Behringwerke AG, Marburg Labs, W. Germany. Macrophage monolayers were incubated with neuraminidase (50 U/ml in PBS) for 3tiO min at 37”C, and washed with PBS (2x2 ml). Erythrocyte suspensions (5X lO’/ml) were treated with neuraminidase (40 U/ml) for 30 min at 37°C and washed twice with PBS.
Yeast cells Yeast cells, Saccharompces cerevisiae, from stationary slants kept at 4°C. Exp CellRes 103 (1976)
Lectin-mediated interaction of erythrocytes and macrophages was brought about in two stages: (a) macrophage-lectin interaction; (b) erythrocyte interaction with lectincoated macrophages. The characteristics of lectin-mediated erythrocyte-macrophage interaction was assessed in parallel for ConA and WGAmediated interactions. The number of erythrocytes attached, at 24”C, to either macrophages coated with ConA (M-ConA) or to macrophages coated with WGA (MWGA) depends on the concentration of erythrocytes incubated with the macrophage monolayers (fig. 1). In order to be able to follow subtle differences in cell-cell interaction patterns mediated by both lectins, erythrocytemacrophage interaction was always assessed at densities of l-2 x 10’ erythrocytes/ ml. The kinetics at 37°C of lectin mediated cell-cell interaction reveals that throughout the 60 min incubation the rate of erythrocyte attachment to M-ConA is constant (fig. 2A). The lag of 5 min in the establishment
were taken
OL 5
,” :::li.:-4 15 20
25
33
35
temp. (“C); ordinate: no. erythrocytes attached per 100macrophages. Temperature dependence of ConA-mediated erythrocyte macrophage interaction. Macrophages were coated with 5 pg/ml (O-O); 10 pglml (A-A); and 40 pg/ml (&-A) of ConA (14°C. 30 min). Subsequent to X2 wash in PBS, macrophage monolayers were incubated with 10’ erythrocytes/ml, for 60 min at the specified temperatures.
Fig. 3. Abscissa:
Differential lectin effects in erz!throphagocytosis B
1 2c
I
I
I
I
’
25
30
35
40
temp. (“C); or&are: (lej?) no. erythrocytes associated with 100 macrophages: (right) (B) 7% erythrocyte ingestion out of total macrophageassociated erythrocytes; (C) no. erythrocytes ingested per 100 macrophages. Temperature dependence of WGA-mediated erythrocyte macrophage interaction. Macrophages were coated with 2 pg/ml (0-O); 5 pg/ml (O-O); 10 pg/ ml (A-A); and 40 pg/ml (A-A) of WGA (14”C, 30 min). Subsequent to X2 wash in PBS. macrophage monolayers were incubated with 10’ erythrocyteslml for 60 min at the specified temperatures.
Fig. 4. Abscissa:
of cell-cell connections is probably due to the fact that erythrocyte suspensions were initially at a temperature of 20°C and ConAmediated interactions are highly temperature-sensitive (see fig. 3). The total number of attached erythrocytes exceeds by approx. 2.5-fold that observed under comparable conditions at 24°C (fig. 1). No ingestion of attached erythrocytes could be detected even after 60 min at 37°C (fig. 2A). The interaction of erythrocytes with MWGA (fig. 2B) reaches saturation within the 60 min incubation, a maximum value being reached already at 30 min. Of the interacting erythrocytes an increasing proportion is ingested with time. After a lag period of about 10 min ingestion increases until it reaches a value of 45% at 60 min. The phase difference between the attachment stage and ingestion stage reflects timedependent processes that are a prerequisite for ingestion to take place. This is even more obvious upon dissociation of attach-
283
ment from ingestion by preformation of rosettes at 24°C and then after ~2 wash in PBS incubation of the rosettes at 37°C (fig. 2B). Again a lag period is observed before the onset of ingestion, and the fraction of ingested erythrocytes does not exceed 30 %. A comparison of the number of erythrocytes associated with macrophages at 24°C (2x lo7 erythrocytes/ml) (fig. lj and 37°C (fig. 2A, B) at the end of a 60 min incubation reveals that whereas in the case of [he ConA-mediated interaction the number of attached erythrocytes increases from 200 to about 500, the number of macrophage associated erythrocytes in the WGA-mediate interaction is not affected by the change of temperature. A detailed analysis of the temperature dependence of the interaction is given En figs 3 and 4. Macrophages were treated with different concentrations of ConA, i.e. 5, LO and 40 pg/ml (fig. 3). At the three concentrations tested a non-linear high temperature dependence of interaction was observed with a steep increase in celL-ceBI association in the temperature ranges of 8-14°C and 19-3X. At 5 pg ConAIm the interaction was practically nil below 25°C.
I”- IO 5
15
20‘5
30
35
40
Fig. 5. Abscissa: temp. (“C) of macrophage coating with ConA; ordinare: no. erythrocytes attached to IO0 macrophages Dependence of erythrocyte-macrophage ConAmediated interaction on the temperature at which ConA coating of macrophage was performed. Macrophages werecoated with 2gpglrnl konA at the specitied temperatures. Subsequent to x3 wash in PBS. ConA coated macrophagei were incubated with 10’ erythrocytes/ml for 60 min at 14°C (O-O); 18°C (A-A); 24°C (O-O): and 37°C (A-A),
284
Goldman and Bursuker
Fig.
6. Electron micrographs of ConA and WGAmediated attachment of erythrocytes to macrophages. Macrophages were coated with 20 pg/rnl of either WGA (a, b) or ConA (c, d) for 30 min at 14°C. Subsequent to ~2 wash in PBS macrophage monolayers were incubated with 10’ erythrocytes/ml at (a, b, d)
19°C and (c) at 30°C. Note the very tight interaction and continuous contact at membrane-membrane apposition when attachment is bridged by WGA (a, b) as opposed to the looser contact when attachment is mediated by ConA (c, d). a, c, X 16900; b, d, X20800.
The concentration of WGA at which maximal erythrocyte-macrophage interaction is established is remarkably lower than that required for reaching an optimum in the ConA-mediated interaction. At 37°C
2 pg WGA/ml suffice to establish the same number of stable cell-cell associations as those brought about by 5, 10 and 40 ,ug/ml (fig. 4A). The temperature at which cell-cell as-
Exp CellRes 103(1976)
Diffeerential lectin effects in erythrophagocytosis sociation is brought about is much less critical in the case of WGA-mediated interaction than with ConA-mediated association. At 2 pg WGA/ml there is a linear dependence of cell-cell interaction with temperature (fig. 4A). At 5, 10 and 40 pg WGA/ml6688 % of the interaction at 37°C was already observed at 8°C. A significant proportion of ingestion of attached erythrocytes was observed only at 30°C and 37°C and the higher the concentration of WGA in the macrophage treatment, the higher was the percent of ingested erythrocytes (fig. 4B, C). Macrophage interaction with ConA and WGA results in cross-linking of surface glycoproteins and cluster formation [7]. Lectin-macrophage interaction as reflected in both the amount of bound lectin, and its subsequent internalization is temperature dependent. The effect of temperature at which lectins were bound to macrophages on the subsequent attachment of erythrocytes, at different temperatures, was therefore studied. Fig. 5 shows the effect of exposure of macrophage monolayers to ConA at different temperatures on a subsequent erythrocyte attachment at 14, 18.5, 24 and 37°C. The highest degree of erythrocyte attachment obtained at any of the temperatures applied was that following a ConA treatment stage at 14 or 18S”C. The highest sensitivity to the temperature of lectin treatment was observed at a temperature of attachment of 14°C. Allowing for extensive cluster formation of ConA-glycoprotein conjugates at 24°C and 37°C reduces the chances for a subsequent erythrocyte attachment at 14°C to 10% and 2 % of maximal attachment, respectively. When cell-cell interaction is brought about at 24°C and 37”C, erythrocyte attachment is only slightly lower than that observed at 14-18.5”C. The data in fig. 5 further indicate the high temperature
285
dependence of ConA-mediated attachment i.e. 3-5-fold higher at 37°C than at any of the other temperatures at which the association was assessed. An analogous set of experiments to that represented in fig. 5 with WGA-mediated erythrocyte attachment indicates non-significant differences in attachment at any of the temperatures subsequent to macrophage treatment with WGA at 8, 14, 18.5, 24 and 37°C. A trend for a maximal attachment following WGA binding at 18.5”C was observed. Erythrocyte interaction with WGAtreated macrophages at 37°C results in a 40% ingestion of the interacting erythrocytes within 60 min. This is true for WGA interaction with macrophages at 8. 24. 18.5 and 24°C. When macrophages were exposed to WGA at 37°C the subsequent interiorization of attached erythrocytes decreased to 10% (100 % being 590 erythrocytes attached at 37”C/lOO macrophages). The difference in the characteristics of ConA- and WGA-mediated erythrocyte attachment to macrophages was further assessed by attempts to form rosettes under conditions of mild rotation or rocking. Under conditions of rotatory motion in the suspension (80 rotations/min) essentially no stable contact between erythrocytes and ConA- orWGA-treated macrophages was established (30 min, 37°C). A rocking motion (17 complete cycles of upward and downward motionsimin) sufficed to reduce to nli and to 30% erythrocyte interaction with ConA- and WGA-treated macrophages. respectively. Of the 210 erythrocytes associated with 100 M-WGA, 97 were ingested within the 30 min of incubation. Control values of interaction under nondisturbed exposure of erythrocyte suspensions to lectin-treated macrophages are given in fig. 2 (30 min). At this point it should be emphasized that the wash condi-
286
Goldman and Bursuker
tions after rosette formation consist of two periods of rotatory shaking in PBS. Thus, all attached erythrocytes are those that establish cell-cell contact withstanding the shear force of the rotatory motion. Electron micrographs of ConA- and WGA-mediated erythrocyte attachment to macrophages are given in fig. 6. Erythrocyte membranes show very extended and extremely tight interaction with WGAtreated macrophages at a temperature of interaction as low as 19°C (fig. 6a, b). A distinctly looser interaction is observed in the ConA-mediated attachment even when the interaction is brought about at 30°C (fig. 6~). At 19°C except for rare occasions (fig. 6d) the gaps between erythrocytes and macrophages are so wide that no certainty exists regarding a real interaction. This is most probably due to the fact that small areas of contact develop and while sectioning the chance to observe these areas is scant. A very tight interaction is also observed between erythrocytes attached to WGA-treated macrophages. A small section of two adjacent erythrocytes can be observed in fig. 6a. While counting erythrocytes associated with M-WGA, erythrocyteerythrocyte associations at the periphery were often observed. Clusters of erythrocytes are rarely ingested and are often the cause of the observation that ingestion does not exceed 40-50 %. It was also established that agglutination of erythrocytes via WGA does not lend itself to ready resuspension, and single cell suspensions could therefore not be obtained. This is the reason that erythrocytes coated with WGA were not amenable to parallel studies to those performed with ConA-coated erythrocytes [4]. WGA-mediated erythrocyte attachment to macrophages at 22°C was 80-90% reversible upon interaction with N-acetyl-nglucosamine (0.1 M, 30 min, 22°C) up to 3 h Exp Cell Res 103 (1976)
of interaction. At 6 h interaction at 22°C or 24 h at 19” only 70% and 35 %, respectively, could be detached by treatment with the inhibitor . During the enumeration of hundreds of macrophage-erythrocyte rosettes generated under the different conditions specified in the figures it became clear that ConA- and WGA-mediated cell-cell interaction differs in yet another aspect. While in the ConAmediated erythrocyte-macrophage interactions the erythrocytes were always localized at the very distant periphery of the cell, those attached via WGA assumed various locations, from peripheral at low temperatures or short interaction times to perinuclear at longer times of incubation at temperatures of 324°C. It was also notable that all the ingested erythrocytes assumed a distinct perinuclear position. In order to follow possible changes in erythrocyte location on the macrophage subsequent to its primary attachment, preformed rosettes (14”C, 30 min) were observed under phase contrast microscopy in a Nikon M microscope equipped with an incubator, set at 37°C. Single cells were followed for 30-60 min from the time of the temperature shift to 37°C and photographed at 3 min intervals. Fig. 7 represents 3 time points out of a sequence of pictures that were taken for (i) ConA-mediated and (ii) WGA-mediated rosette formation. The Fig. 7. Rel. movement of erythrocytes and yeast cells attached to macrophages via lectins. Macrophages were treated with either 20 pg/ml (a-c, g-i) ConA; or (d-f) WGA at 14”Cfor 30 min. Subsequent to x2 wash in PBS macrophage monolayers were incubated with 10’ (u-f) erythrocytes or (g<) yeast cells for 30 min at 14°C. Preformed rosettes were immediately washed in PBS. transferred to fresh PBS and incubated at 37°C for continuous microscopic observation. The time sequence represented in the figure is, macrophage-ConA-erythrocyte: (a) 10 min; (b) 20 min; (c) 25 min; macrophage-WGA-erythrocytes: (d) 10 min; (e) 23 min; v) 30 min; macrophage-Con&yeast cells: (g) 5 min; (h) 11 min; (i) 21 min. X 1320.
Differential
lectin effects in erythrophagocytosis
2
288
Goldman and Bursuker
Table 1. SBA-mediated Macrophage treatment Neuraminidase * + + + + + + +
SBAC (pdml) J 5 40 40 -
erythrocyte
macrophagea
interaction
Erythrocyte treatment Neuraminidaseb
SBAC Q4h-4
+ + + + + +
2 2
No. attached erythrocytesllO0 macrophages 0 22 3 3 20 35 104 158 188
a Erythrocyte suspensions (7.5X lo6 cells/ml) were incubated with macrophages for 60 min at 22°C. b Neuraminidase treatment as described in Methods. c SBA treatment of macrophages and erythrocytes, 30 min, 22°C. Erythrocyte suspensions 75x lo6 cells/ml.
two erythrocytes seen in fig. 7a-c are attached at the distant periphery of the macrophage membrane. No changes in the spatial relation of the erythrocytes to the peripheral regions or to the nucleus were observed during the 25 min incubation at 37°C. At the same time, phase lucent vesicles derived from ConA pinosome fusion were generated [7, 111. Fig. 7d-f represents the sequence of erythrocyte movement subsequent to attachment to a WGAtreated macrophage. At 10 min, two erythrocytes were still located at a peripheral position, three of the other erythrocytes had a different plane of focus. At 23 min incubation at 37°C the two erythrocytes changed their position relatively to both the border of the cell and the nucleus. At 30 min four erythrocytes had a common plane of focus and the distances between them had decreased. From the above it is impossible to decide whether the erythrocytes move or whether the macrophage spreads over their surface in the undulating movement of the membrane and the whole segment of the interacting membrane with the attached erythrocyte assumes a more central position relative to the nucleus. Exp Cell Res 103 (1976)
The interaction of M-ConA with yeast cells has been characterised in some detail [5]. In the case of ConA-mediated attachment of yeast cells the attachment leads to yeast interiorization in contrast to erythrocyte behaviour when attached via ConA but conforming with erythrocyte ingestion when attachment is mediated by WGA. In order to assess whether the centripetal movement is a prerequisite for phagocytosis and whether ConA-treated macrophages are not distinct in this respect from WGAtreated macrophages, the dynamics of interaction of yeast cells with ConA-treated macrophages was followed. Fig. 7 (g-i) represents the interaction of preformed yeast cell macrophage rosettes (30 min, 14°C) at 5, 11 and 21 min subsequent to transfer to 37°C. The yeast cells can readily be seen to change their position relative to the periphery, nucleus and to each other. Under these conditions 70% of attached yeast cells are ingested during a 60 min incubation [S]. Erythrocytes coated with SBA, though readily agglutinable, failed to establish stable rosettes with macrophages. Recently it has been observed that SBA interaction with macrophages requires a first step of
Differential lectin effects in erythrophagocytosis
289
Table 2. A rough quantitative estimate of random surface distribution of ConA and VGA
Cell type
(pm?)
Ref.
No. bound ConA moleculeslcell (X 1OP) Ref.
Macrophage Erythrocyte Yeast ceil
2 500”
[24]
5
Surface area
50 60
[71
1 [51
3.4
Surface density (ConA/pm”)
No. bound WGA molecules/cell (X 10-y Ref.
2 wo
15”
20 000 57 000
3’ N.D.
E71
Surface density CNGAl~nPi 6 ooo 50000 N.D.
’ A minimum value [24] that might need a reconsideration in view of the many ruffles seen in scanning elecl;ron microscopy [23]. * A minimum value based on an estimate of 50% lectin interiorization. A three-fold value of WGA binding over ConA binding was obtained for SV40-transformed golden hamster cells [14]. ’ .4 three-fold value of ConA binding was taken as a first approximation.
neuraminidase treatment and that polymerized SBA binds also to non-treated macrophages [7]. The ability of SBA to mediate erythrocyte-macrophage interaction was therefore tested in combination with neuraminidase treatment of either one or both cell types. Table 1 indicates that a significant interaction via SBA requires a pretreatment of erythrocytes with neuraminidase. The mild vacuolation observed in macrophages treated with 40 pg/ml SBA implies that a certain fraction of the SBA was in an aggregated state [7]. When neuraminidase-treated erythrocytes were coated with SBA and incubated with macrophages the latter established stable association with the erythrocytes whether they were pretreated with neuraminidase or not. Transferring preformed rosettes to 37°C resulted in total dissociation of erythrocytes from macrophages. This is in line with a lower association constant of SBA to glycoproteins at 37°C than at 22°C. WBA-coated macrophages (30 pg/ml, 30 min. 22°C) interact with erythrocytes (10’ cellsiml, 60 min, 22°C) to yield rosettes consisting of .547&40 erythrocytes/lOO macrophages. Incubation of preformed rosettes at 37°C in medium for 90 min did not result in a detectable ingestion of attached erythro-
cytes. The number of attached erythrocytes was not reduced, however, during the additional incubation period. DISCUSSION The interaction of erythrocytes with macrophages coated with ConA or WGA results in the formation of stable cell-cell associations. “Stable” means in the present experimental design an association that withstands two steps of 20 set wash in PBS on a rotatory shaker operated at a speed of 100 rotations/min. At early phases of cell-cell contact the shear forces developing in the solution by the rotatory motion do not allow for stable associations, i.e. when the interacting cells are incubated together under rotatory shaking conditions rosette formation is precluded. Rosette formation strong ly depends on the concentration of ery-throcyte suspensions. At 5 x 10’ erythrocytes! ml, ConA-mediated rosette formation is far from saturation, reaching a value of 550 erythrocytes/ 100 macrophages. The interaction via WGA is more efficient, resulting in the association of about 1300 erytbrocytes/IOO macrophages (fig. 1). The time dependence of rosette formation at 37°C (fig. 2) further establishes the
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Goldman and Bursuker
difference in rates at which the interaction via ConA and WGA approaches saturation. Since the number of collisions of erythrocytes and the macrophage monolayer is the same whether macrophages were coated with ConA or WGA the different rate must reflect a differential development of strength of binding. Moreover, the WGAmediated erythrocyte attachment to macrophages can, after a certain lag period, trigger erythrophagocytosis (fig. 2B). Thus the more efficient binding is followed by a subsequent step of phagocytosis. The lag period between attachment and ingestion could reflect the need for a circumferential attachment of the plasma membrane around the erythrocyte. If one regards the process of phagocytosis as the spreading of the macrophage plasma membrane around a particle analogously to its spreading on glass [13] then it is reasonable that the rate of spreading would reflect the affinity between the two surfaces, that of the macrophage and that of the particle. Most interesting is the observation that ConA-coated macrophages fail to complete the encircling of the attached erythrocyte while when coated with WGA they do so with a good efficacy; i.e. 40% of the attached erythrocytes are phagocytosed within 1 h. WBA and SBA are also capable of serving as a bridge for rosette formation, however, neither triggers erythrophagocytosis. For SBA mediates rosette formation it is essential that the erythrocytes be pretreated with neuraminidase. Macrophage pretreatment with neuraminidase is less critical for rosette formation though it increases the interaction (table 2). ConA-mediated erythrocyte attachment (hemadsorption) has a non-linear dependence on temperature with a 3-fold increase in the range of 1837°C. This is true at three different degrees of saturation of ConAExp Cell Res 103 11976)
binding sites on the macrophages. A similar pattern has been observed for the ConAmediated agglutination of suspended SV40transformed hamster cells [lo, Ill. In contrast Rittenhouse & Fox [ 151,studying hemadsorption to ConA-coated LM cells, observed a very sharp temperature effect, resembling a phase transition in the range of 12-18°C below which hemadsorption amounted to about 10% of that observed at 18°C and above which no further increase in hemadsorption has been observed. The sharp transition range could be shifted to a lower and a higher temperature range depending on the composition of fatty acids in membrane phospholipids [16]. ConAmediated yeast cell-macrophage association [5] has a less pronounced temperature dependence; 60-70% of the yeast cells attached to macrophages at 25°C were already attached at 15°C and no increase in attachment was observed in the range of 25-37°C. The difference in the temperature dependence of ConA-mediated attachment of erythrocytes and yeast cells brings up the contribution of ConA-binding sites on the counter cell to the interaction pattern. WGA-mediated hemadsorption is much less temperature dependent than that of the ConA-mediated hemadsorption (fig. 4). Macrophages precoated with 5, 10 and 40 pg WGA/ml interact to a similar degree with erythrocytes at 8-37°C. Precoating of macrophages with 2 pg WGA/ml results in sensitized cells with a hemadsorption capacity that is extremely temperature sensitive. It is possible that cell-bound WGA is distributed very sparsely on the membrane and lectin receptor conjugates or small crosslinked clusters have to be aggregated at the area of apposition of two membranes for development of stable contacts. The lateral mobility of membrane glycoproteins increases with temperature [ 17, 181.It is note-
Differential lectin effects in erythrophagocytasis worthy that at 37°C hemadsorption to macrophages coated with a solution of 2 pg WGAlml reaches the level of maximal hemadsorption achieved under conditions of higher saturation of lectin-binding sites on the macrophage membrane (fig. 4). The ingestion efficiency of macrophages for attached erythrocytes does, however, decrease in rosettes formed with macrophages of a lower degree of saturation in WGAbinding sites (fig. 4B, C). In comparing the degree of hemadsorption achieved by precoating macrophages with WGA and ConA at various concentrations it is important to take into account the different physical parameters of both lectins. ConA and WGA differ markedly in their molecular dimensions (102000 and 36000 D mol. wt, respectively [19-201) though both possess four sugar-binding sites. Macrophage-binding sites for WGA are more abundant than those for ConA as was also observed for other cell types [6, 7, lo]. It is interesting that Vlodavsky & Sachs [lo] report a higher apparent association constant for membrane glycoproteins for ConA (lo7 M-l) than for WGA (1.6~ IO6M-l). The striking difference between the pattern of rosette formation and the fate of attached erythrocytes is fully expressed even when the compared conditions consist of 5 pg/ml of WGA and 40 pg/ml of ConA in the coating so&ion. ConA-mediated cluster formation of glycoproteins requires a short range lateral movement of the latter compounds. Once they are clustered and cross-linked by ConA their lateral diffusion is much hindered [I7]. Both processes, initial cluster formation and redistribution of cluster components are temperature dependent, It is only plausible that the redistribution of glycoprotein-receptor conjugates will be
291
more sensitive to temperature than that of the non-cross-linked glycoproteins. Pectin coating of macrophages at different temperatures results in various degrees of cluster formation as well as in interiorization of lectin receptor conjugates [7]. When the interaction of ConA with macrophages is carried out at 2537°C the clustering is such that a shift to 14°C for a period of incubation with erythrocytes does not allow for extensive redistribution of receptors and extensive hemadsorption. If incubation with erythrocytes is carried out at 24°C or better still, at 37”C, redistribution of clusters allows for hemadsorption amounting to 70% of maximal observed hemadsorption, The highest value of hemadsorption is obtained following a lectin-coating step carried out at 14-18°C. The physical state of membrane phospholipids appears to be highly sensitive to variations in this temperature range [ 181. Hemadsorption mediated by WGA is much less influenced by the temperature at which macrophages are pretreated with the lectin. The characteristic differences in ConAand WGA-mediated hemadsorption are in line with those observed for lectin-mediated agglutination of SV40-transformed hamster cells [lo, 141and for agglutination of erythrocytes [S, 91. Mechanical shear introduced by shaking interferes strongly with ConAmediated erythrocyte agglutination, but has only a small effect on WGA-mediated agglutination [$I. The data reported above indicates a higher resistance to shear forces of WGA-mediated erythrocyte-macrophage rosettes than that of ConA-mediated rosettes. Sachs et al. [lo, 14, 211 propose that ConA-binding sites have a high short range lateral mobility that has to be restricted before effective agglutination or ceil binding to ConA-coated nylon fibres takes place.
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According to this criteria the more abundant WGA-binding sites have a much lower lateral mobility and therefore WGAmediated agglutination or attachment to fibres is much less temperature sensitive [IO, 14,211. ConA- and WGA-binding sites may reside on different glycoproteins or at least the overlap in receptors for either lectin may involve a small fraction of the binding entities [6]. In contrast to the manifold data regarding the factors governing the surface distribution of ConA-binding sites the data about the lateral mobility of WGA-binding sites is rather scant. On the basis of the above discussed facts, inferences and a rough estimate of the quantitative parameters involved (table 2), the following scheme is suggested for the differential effect of ConA and WGA on erythrocyte attachment and ingestion. Phagocytosis requires a multibridge interaction along the entire circumference of the particle. When lectin molecules bound to macrophages serve as a bridge the primary attachment area as well as the membrane enveloping the particle have to contain lectin receptor conjugates. Under conditions of extensive clustering of lectin receptor conjugates as those implied to develop in ConA-treated macrophages the plasma membrane surrounding the area of erythrocyte attachment may be deprived of, or poor in, free lectin-binding sites and as a result no stable circumferential interaction can be established. Since not all of the ConA-binding sites are saturated under the experimental conditions, addition of ConA to preformed rosettes that do not undergo erythrocyte ingestion triggers full erythrophagocytosis of attached erythrocytes [4]. The additional ConA may coat both the macrophages as well as the erythrocytes. WGA-binding sites are more abundant on Exp Cell Res 103 (1976)
the macrophage than ConA-binding sites. If in addition they exhibit lower tendency for cluster formation [IO, 14, 211 then WGA distribution on the macrophage plasma membrane may enable both the interaction with erythrocytes leading to attachment and the subsequent interaction of adjacent plasma membrane to achieve circumferential interaction. A very tight interaction and membrane “crawling” to envelop erythrocytes attached to macrophages via WGA is indicated in fig. 6. It should be kept in mind that due to the high mobility of surface receptors many receptors can be aggregated at interaction sites with the particle. Taking a value of 1.5~ low9 cm2/sec for the diffusion constant of surface macromolecules [17] then a glycoprotein molecule can cover a distance of 6 ,u/min. Edidin & Fambrough [17] have shown that cross-linking of surface macromolecules labelled with Fab by an antiFab immunoglobulin reduces the rate of surface spread of Fab. They have estimated a reduction by a factor of 10 (0.15~ lops cm2/ set) in the diffusion constant. In analogy therefore it is expected that cross-linking of glycoprotein receptors via ConA will reduce their mobility by a factor of -3 (X2= 4 Dt). The estimated mobility of a surface cross-linked macromolecule is still very high relative to cellular distances, and cluster formation should therefore be regarded as a dynamic process; in a time scale of minutes small clusters should readily change place and reorganize unless crosslinked to an external particle (yeast, erythrocyte). Big clusters (patches) induced by polyvalent lectins may have a different, larger, time factor for spreading and reorganization. Erythrocytes tend to establish the primary contact with the macrophage at cell peripheral regions where the undulating membrane is flat and well spread out on the
Differential lectin effects in erythrophagoqvtosk
293
glass. This is true for both ConA- and teriorization is clearly suggested from the WGA-mediated attachment. When attach- differential behaviour of ConA-mediated ment is not followed by an ingestion step yeast cell and erythrocyte-macrophage ro(ConA-mediated erythrocyte-macrophage settes. The density of ConA-binding sites rosettes) the erythrocytes do not change on mouse erythrocytes is about 3-fold lower than that observed for yeast ce!ls (table 2) their position during prolonged incubation periods. Attachment that is followed by an where a closely packed monolayer of ConA ingestion step (WGA-mediated macro- is accommodated at ConA saturation ]:5]. phage-erythrocyte rosettes and ConA- Thus, under comparable conditions of macmediated macrophage-yeast cell rosettes) rophages coating with ConA, the yeast cell results in a subsequent movement of the is more efficient than erythrocytes in reattached cells to a perinuclear position and cruiting ConA receptor conjugates and in only at this position phagocytosis seems to establishing stable circumferential intertake place. It is impossible to decide at this actions. As a result, yeast cells are ingested stage whether the erythrocyte moves or with very high efficiency while the erythrowhether it is encompassed by the plasma cytes are not. It is interesting to note that membrane at distal edges and then the yeast cells attached to macrophages whole complex of undulating membrane en- changed their position on the macrophage veloping the erythrocyte moves towards with time much the same as erythrocytes the centre. The latter possibility is more attached via WGA (compare fig. 7g-i with plausible. Membrane envelopment of 7d-j). erythrocyte at distal regions of the macrophages prior to phagocytosis have been obREFERENCES served using scanning electron microscopy 1. Mantovani, B, Rabinovitch. M & Nussenzweig, Z, [22]. Another possible mechanism that was J exp med 135 (1972) 780. 2. Griffin, F M. Jr & Silverstein, S C. J exp med 139 considered is the formation of craters (1974) 323. through which particles are ingested. Kap3. Griffin, F M, Jr, Griffin, J A, Letder, J E & Silverstein, S C, J exp med 142(1975) I263. lan et al. [22] suggest that this mechanism is 4. Goldman, R & Cooper. R A. Exp celires 95 (1975) restricted to small phagocytosible particles 223. 5. Bar-Shavit, Z & Goldman, R. Exp csii res 99 and that this phagocytosis is mostly peri(1976) 221. nuclear. Polliack & Gordon [23] have ob- 6. Nicolson, G L, Int rev cytol 39 (1974) 89. 7. Goldman, R, Sharon. N & Lotan, R, Exp cei! res served crater formation during the inter99 (1976) 408. action of macrophages with erythrocytes. 8. Schnebli, H P & Bachi. T, Exp cell res ?! (1975) 175. Accepting either model it is clear that 9. Vlodavsky, I, Inbar, M & Sachs, L, Biochim biotriggering the steps following attachment be phys acta 274 (1972) 364. it membrane crawling around the particle or IO. Vlodavsky, I & Sachs. L, Exp cell res 9; (1975) 202. the formation of craters due to sinking-in of II. Goldman, R & Raz, A, Exp cell res 96 (1975) 393. 12. Spurr, A R, J ultrascruct res 26 (1969) 3 1. the membrane would depend on additional Stossel. T P, Seminars in hematol 12 (1975) 83. interaction between the particle and the 13. 14. Vlodavsky, I & Sachs. L. Exp cell res 93 (1975) 111. plasma membrane. These events would be H G & Fox, C F, Biochem biophys both temperature and time dependent (figs 1.5. Rittenhouse, i-es commun 57 ( 1974)323, 16. Rittenhouse. H G. Williams. R E & Fox, C F, J 2,4: 7). supramol struct 2 (1974) 629. The importance of the characteristics of 17. Edidin, M & Fambrough, D, J ceh bio? 57 (1973) 27. the ingested particles in triggering the in-
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18. Petit, V A & Edidin, M, Science 184 (1974) 1183. 19. Reeke, G N, Becker, J W, Cunningham, B A, Gunther, G R, Wang, J L & Edelman, G M, Ann NY acad sci 234 (1974) 369. 20. Nagata, Y &Burger, M M, J biol them 249 (1974) 3116. 21. Rutishauser, U & Sachs, L, J cell biol66 (1975) 76.
15x-pCell Res 103 (1976)
22. Kaplan, G, Gandernack, G & Seljelid, R, Exp cell res 95 (1975) 365. 23. Polliack, A&Gordon, S, Lab invest 33 (1975)469. 24. Werb, Z &Cohn, Z A, J exp med 134(1971) 1570. Received June 9, 1976 Accepted June 11, 1976
DIFFERENTIAL
EFFECTS
ERYTHROCYTE
OF LECTINS
ATTACHMENT
AND
MEDIATING INGESTION
BY MACROPHAGES RACHEL GOLDMAN Department
of Membrane
Research,
and I. BURSUKER
Weiynann
Institlrre
of Science, Rehovor, Israel
SUMMARY Lectin-mediated interaction of erythrocytes and macrophages was brought about in two steps. Step I involved macrophage treatment with lectin. and step II is the incubation of lectin-treated macrophages with mouse erythrocytes. The extent and nature of lectin-mediated macrophage erythrocyte interaction was studied using concanavalin A (ConA), wheat germ (WGA), soybean (SBA) and waxbean (WBA) agglutinins. The parameters affecting the interaction were studied in detail with the first two lectins. Under comparable conditions of lectin interaction with macrophages (step I). WGA mediates rosette formation involving interaction with several times the number of erythrocytes than those interacting with ConA-treated macrophages. The interaction mediated by WGA reaches, at 37°C. a saturation value after 30 min of step II. whereas that mediated by ConA is still linear and exhibits half the amount of attached erythrocytes at 60 min. Cot&mediated attachment of erythrocytes is highly temperature-dependent being at 37°C twice that observed at 24°C. The temperature dependence of attachment is not affected by changes of either ConA concentration (5-40 pg/ml) or the temperature in step I. An optimum is observed, however. when the temperature of incubation in step I ranges between 14-18°C. WGA-mediated attachment of erythrocytes is markedly less temperaturesensitive, exhibiting 70% of optimal attachment already at 8°C. Only when the attachment phase foliows incubation with a low concentration of WGA (2 pg/ml) high temperature sensitivity is exhibited. At 3?“C, however, the number of attached erythrocytes is the same for macrophages treated with WGA at concentrations of 2. 5, 10 and 40 pg/rnI. ConA-mediated erythrocyte-macrophage interaction does not lead to erythrophagocytosis. When mediated by WGA, the attachment step is followed by a temperature-dependent ingestion step. i.e. 10% and 50% of the erythrocytes that attach to macrophages during the 60 min incubation at 24°C and 37°C. respectively. are ingested. There is a lag period of 10-20 min between attachment and ingestion implicating involvement of additional cellular processes preceding engulfment. Electron microscope images of areas of interactron of attached erythrocytes with macrophages indicate a significantly tighter binding (a thinner gap at membrane-membrane apposition areas) in the case of WGA-mediated rosette formation as compared with that established in ConA-mediated rosettes. Attachment via WGA is followed by a rapid change in the re!ative position of the attached erythrocytes on the macrophage, from a primary attachment at the distal peripheral regions of the cell, to a perinuclear position. In contrast, erythrocytes attached via ConA remain at the primary attachment point (at 37°C) for extended periods. This differential behaviour does not stem from effects of ConA on macrophages, since when yeast cells were attached to ConA treated macrophages. the yeast cells showed the same movement as that exhibited by erythrocyte when attached via WGA. The different interaction patterns of erythrocytes with macrophages coated with ConA and WGA can be fit:cd into the following working hypothesis: the number of WGA-binding sites on the plasma membrane of macrophages is at least three times that of ConA-binding sites. Stable cell-cell interactions involve multibridge formation at the contact area of the two cells and this involves a delicate balance between number of iectin-receptor conjugates and their aggregation state within the membrane phase. A certain amount of clustering is a prerequ;site for attachment, while a high degree of clustering reduces the chance of fruitful interactions. The engulfment step depends on the ability of membrane areas adjacent to primary contact area to establish additional stable bridges in the entire circumference of the attached cell. ConA-receptor conjugates appear to be less abundant and more aggregated within the membrane plane. preventing the completion of fruitful circumferential interaction of the adjacent membrane. WGA-receptor conjugates, being more abundant and apparently less aggregated are available at membrane areas needed for cell enclosure and provide the additional bridging without which engulfment does not take place. Change in relative position of attached erythrocytes seems to be a step in the manifold events occurring from attachment to ingestion.
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The means by which phagocytes recognize and ingest have been a long-term pursuit in diverse fields. The mechanism for recognition and attachment is generally divided into two main categories, immune and nonimmune recognition. In spite of the extensive studies, the process still poses many riddles to the investigator. One of the most exciting questions is the way in which contact could trigger the events leading to formation of phagosomes and the molecular organizational events on the membrane level that precede it. A number of studies have established that though attachment of particles is a prerequisite for their ingestion by macrophages, attachment per se is not a sufficient signal for particle interiorization. In immune phagocytosis it was established that the attachment process is distinct from the ingestion process in both temperature dependence [l] and the degree of required opsonization, be it with IgG alone or IgG+ the C3 component of complement [ 11.It has been shown that the process is specific and limited to the segments of plasma membrane to which the ingested particles are bound [2] and that ingestion requires the sequential, circumferential interaction of particle bound ligands (IgG, IgM+CJ with specific plasma membrane receptors not involved in the initial attachment process [3]. Previous work in our laboratory has characterized a concanavalin A (ConA)mediated attachment and ingestion of fresh homologous erythrocytes and yeast cells with mouse peritoneal macrophages [4, 51. Erythrocyte- or yeast cell-macrophage rosettes were formed under conditions in which either the erythrocytes or yeast cells were ConA coated or the macrophages were ConA coated. The attachment of ConA-treated erythrocytes to macrophages was more efficient than the attachment of Exp Cell Res 103 f 1976)
erythrocytes to ConA-treated macrophages (M-ConA). In neither case, however, was the attachment followed by erythrocyte ingestion. Addition of ConA to preformed rosettes and further incubation at 37°C resulted in 100% ingestion of the attached erythrocytes [4]. It was argued that the additional ConA was needed for saturation of ConA-binding sites on the erythrocytes, a saturation that could not be achieved in erythrocyte suspensions. Yeast cells have a higher surface density of ConA-binding sites than erythrocytes [S]. Yeast cell ingestion was triggered in both, rosettes formed from interacting yeast cells coated with ConA with macrophages and in rosettes formed from yeast cells interacting with M-ConA. The extent and temperature dependence of ingestion under the two rosetting schemes, however, showed striking differences [5]. On the basis of the detailed studies concerning ConA-mediated yeast cell-macrophage interaction it was suggested that ingestion involves the recruitment of more ConA bridges at areas of membrane-yeast cell interaction than needed for a stable yeast cell attachment. It was also suggested that the mobility of ConA receptors in the plane of the plasma membrane whether free or ConA-conjugated was an important factor in triggering ingestion, overriding metabolic energy requirements as well as the activity of the cytoskeletal elements (microfilaments and microtubules). Lectin-binding sites in eukaryotic cells represent a heterogeneous population of glycoproteins [6]. Previous work from our laboratory has characterized the differential response of macrophages to various lectins [7]. The agglutination patterns of erythrocytes, and fibroblasts in response to lectin binding has indicated that different lectins
Differential lectin effects in erythrophagocytosis MATERIALS
,.,,i /‘/i
281
AND METHODS
Media Dulbecco’s modified Eagle’s meditim (medium) was supplemented with 100 U/ml penicillin and 100 @g/ml streptomycin, and sterilized by Millipore fiitration. Medium, heat-inactivated new-born calf serum (serum) and (Dulbecco’s) phosphate-buffered saline (!PBSj were obtained from Grand Island Biological Co. ~N.Y.
t //J 500 ?
Lectins and their inhibitors )
/ 2
3
4
5
cont. of erythrocytes suspensions incubated with macrophages, x10-‘; ordinate: no. of erythrocytes attached per 100 macrophages. Lectin-mediated ervthrocvte-macronhage rosette formation. Macrophages were coated wiih either WGA (O-O) or ConA (0-O) (20 fig/ml of lectin. 30 min. 14’C). Subsequent to ~2 ‘wash-in PBS lectin-treated macrophages were incubated with erythrocytes (24”C, 60 min).
Fig. 1. Abscissa:
generate associations differing in their strength [S], concentration and temperature dependency [9, lo]. It was also established that the number of lectin binding sites/cell and the association constants are markedly different [lo]. The experiments reported in this paper were designed to assess the interaction pattern of erythrocytes with lectin-treated macrophages and to find out whether lectinmediated interactions would be able to trigger erythrophagocytosis. The lectins used in the study were ConA, wheat germ (WGN, soybean (SBA) and waxbean (WBA) agglutinins. The analysis concentrated in particular on the contribution of parameters such as temperature, lectin concentration, erythrocyte concentration, and duration of interaction to the formation of erythrocyte-macrophage rosettes, to subsequent erythrocyte ingestion (WGAmediated) and to the spatial arrangement of the erythrocytes relative to the cell periphery and nucleus.
ConA, twice crystallized, was obtained from MilesYeda (Rehovot, Israel) and WGA. WBA and SBA. prepared by affinity chromatography [7], were a gift of Dr R. Lotan. a-Methyl-D-mannopyranoside ((YMM) and N-acetyl-o-glucosamine were obtained from Pfanstiehl Labs. Inc., Waukegan.
Collection and cultivation qf macrophages Peritoneal macrophages were aseptically collected from BALE/c strain male mice, weighing 20-25 g. Peritoneal exudate cells suspended in medium were allowed to attach (5x 10” cells in 0.15 ml. 60 min, 37’9 on either 25 mm diameter Coming cover glasses or cover glasses coated with a thin sheet of parlodion [ll]. The cover glasses were placed in 35 x ld mm Faicon tissue culture dishes (Falcon Plastics Div. Bioquest. Oxnard, Calif.). After the phase of cell attachment the plates were thoroughly rinsed in PBS IO remove non-adhering cells. Macrophages were then cul-tivated for 48 h at 37°C in 2 ml of 20% serum ir. Dulbecco’s modified Eagle’s medium, in a CO,incubator (5 ‘% COTair mixture). Culture medium was changed once after 24 h incubation.
Macrophage interaction uqith iectins Macrophage monolayers were twice washed in PBS and1incubated with the specified lectin at the concentrations, temperature and time specified in the legends to the corresponding figures, and subsequentiy washed twice in PBS.
Erythrocyte-macrophage interaction Freshly obtained mouse blood was anticoagulated iyith citrate phosphate dextrose solution (Travenol Lab. Ltd, Ashdod, Israel), then washed with PBS containing 5 mM of glucose (PBS-glucose) to remove plabma and buffy coat. Unless otherwise specified. 1 ml of an erythrocyte suspension (10’ cells/ml PBS-glucasc) was added to lectin-treated macrophage monoiayers. At the end of the incubation period non-adherent erythrocytes were removed by twice-washing with PBS on a rotatory shaker (100 rotations/min, Clinical Rotator, Eberbach Corp. Ann Arbor. Mich.i. For evaluation of erythrocyte attachment and ingestion, cells were fixed (2% glutaraldehyde in PBS, 30 min at 4°C) and stained (May-GrtiwaldGiemsa).
282
Goldman and Bursuker RESULTS
time (mm); ordinate: (left) no. erythrocytes interacting with 100 macrophages (0-O); (rigkt) % of ingested erythrocytes out of total macrophage associated erythrocytes (O-O). Time dependence of lectin-mediated erythrocytemacrophage interaction. Macrophages were coated (30 min, 14°C. 20 pg/ml) with either (+4) ConA or (B) WGA. O-Cl, WGA-mediated rosettes were formed at 14°C (60 min); washed twice in PBS and transferred to fresh PBS+glucose at 37°C for the specified times; O-0, % ingestion.
Fig. 2. Abscissa:
Erythrocytes attached and ingested per 100 macrophages were enumerated in triplicate cultures. Attached erythrocytes were readily distinguished from ingested erythrocytes by a colour change, glassy appearance and irregular contour of the latter. For electron microscopy cells cultivated on parlodion sheets were allowed to interact with lectins and erythrocytes as specified in the figures. The interacting cells were then processed as described [ 1l] and embedded in epoxy resin according to Spurr [12]. Thin sectioning was obtained with Sorvall, Porter-Blum MT 2-B ultramicrotome and the sections were analysed in a Philips 300 electron microscope operated at 80 kV. The sections were stained prior to visualization with lead citrate.
Treatment of macrophages and erythrocytes with neuraminidase
loot
Neuraminidase from Vibrio comnta was obtained from Behringwerke AG, Marburg Labs, W. Germany. Macrophage monolayers were incubated with neuraminidase (50 U/ml in PBS) for 3tiO min at 37”C, and washed with PBS (2x2 ml). Erythrocyte suspensions (5X lO’/ml) were treated with neuraminidase (40 U/ml) for 30 min at 37°C and washed twice with PBS.
Yeast cells Yeast cells, Saccharompces cerevisiae, from stationary slants kept at 4°C. Exp CellRes 103 (1976)
Lectin-mediated interaction of erythrocytes and macrophages was brought about in two stages: (a) macrophage-lectin interaction; (b) erythrocyte interaction with lectincoated macrophages. The characteristics of lectin-mediated erythrocyte-macrophage interaction was assessed in parallel for ConA and WGAmediated interactions. The number of erythrocytes attached, at 24”C, to either macrophages coated with ConA (M-ConA) or to macrophages coated with WGA (MWGA) depends on the concentration of erythrocytes incubated with the macrophage monolayers (fig. 1). In order to be able to follow subtle differences in cell-cell interaction patterns mediated by both lectins, erythrocytemacrophage interaction was always assessed at densities of l-2 x 10’ erythrocytes/ ml. The kinetics at 37°C of lectin mediated cell-cell interaction reveals that throughout the 60 min incubation the rate of erythrocyte attachment to M-ConA is constant (fig. 2A). The lag of 5 min in the establishment
were taken
OL 5
,” :::li.:-4 15 20
25
33
35
temp. (“C); ordinate: no. erythrocytes attached per 100macrophages. Temperature dependence of ConA-mediated erythrocyte macrophage interaction. Macrophages were coated with 5 pg/ml (O-O); 10 pglml (A-A); and 40 pg/ml (&-A) of ConA (14°C. 30 min). Subsequent to X2 wash in PBS, macrophage monolayers were incubated with 10’ erythrocytes/ml, for 60 min at the specified temperatures.
Fig. 3. Abscissa:
Differential lectin effects in erz!throphagocytosis B
1 2c
I
I
I
I
’
25
30
35
40
temp. (“C); or&are: (lej?) no. erythrocytes associated with 100 macrophages: (right) (B) 7% erythrocyte ingestion out of total macrophageassociated erythrocytes; (C) no. erythrocytes ingested per 100 macrophages. Temperature dependence of WGA-mediated erythrocyte macrophage interaction. Macrophages were coated with 2 pg/ml (0-O); 5 pg/ml (O-O); 10 pg/ ml (A-A); and 40 pg/ml (A-A) of WGA (14”C, 30 min). Subsequent to X2 wash in PBS. macrophage monolayers were incubated with 10’ erythrocyteslml for 60 min at the specified temperatures.
Fig. 4. Abscissa:
of cell-cell connections is probably due to the fact that erythrocyte suspensions were initially at a temperature of 20°C and ConAmediated interactions are highly temperature-sensitive (see fig. 3). The total number of attached erythrocytes exceeds by approx. 2.5-fold that observed under comparable conditions at 24°C (fig. 1). No ingestion of attached erythrocytes could be detected even after 60 min at 37°C (fig. 2A). The interaction of erythrocytes with MWGA (fig. 2B) reaches saturation within the 60 min incubation, a maximum value being reached already at 30 min. Of the interacting erythrocytes an increasing proportion is ingested with time. After a lag period of about 10 min ingestion increases until it reaches a value of 45% at 60 min. The phase difference between the attachment stage and ingestion stage reflects timedependent processes that are a prerequisite for ingestion to take place. This is even more obvious upon dissociation of attach-
283
ment from ingestion by preformation of rosettes at 24°C and then after ~2 wash in PBS incubation of the rosettes at 37°C (fig. 2B). Again a lag period is observed before the onset of ingestion, and the fraction of ingested erythrocytes does not exceed 30 %. A comparison of the number of erythrocytes associated with macrophages at 24°C (2x lo7 erythrocytes/ml) (fig. lj and 37°C (fig. 2A, B) at the end of a 60 min incubation reveals that whereas in the case of [he ConA-mediated interaction the number of attached erythrocytes increases from 200 to about 500, the number of macrophage associated erythrocytes in the WGA-mediate interaction is not affected by the change of temperature. A detailed analysis of the temperature dependence of the interaction is given En figs 3 and 4. Macrophages were treated with different concentrations of ConA, i.e. 5, LO and 40 pg/ml (fig. 3). At the three concentrations tested a non-linear high temperature dependence of interaction was observed with a steep increase in celL-ceBI association in the temperature ranges of 8-14°C and 19-3X. At 5 pg ConAIm the interaction was practically nil below 25°C.
I”- IO 5
15
20‘5
30
35
40
Fig. 5. Abscissa: temp. (“C) of macrophage coating with ConA; ordinare: no. erythrocytes attached to IO0 macrophages Dependence of erythrocyte-macrophage ConAmediated interaction on the temperature at which ConA coating of macrophage was performed. Macrophages werecoated with 2gpglrnl konA at the specitied temperatures. Subsequent to x3 wash in PBS. ConA coated macrophagei were incubated with 10’ erythrocytes/ml for 60 min at 14°C (O-O); 18°C (A-A); 24°C (O-O): and 37°C (A-A),
284
Goldman and Bursuker
Fig.
6. Electron micrographs of ConA and WGAmediated attachment of erythrocytes to macrophages. Macrophages were coated with 20 pg/rnl of either WGA (a, b) or ConA (c, d) for 30 min at 14°C. Subsequent to ~2 wash in PBS macrophage monolayers were incubated with 10’ erythrocytes/ml at (a, b, d)
19°C and (c) at 30°C. Note the very tight interaction and continuous contact at membrane-membrane apposition when attachment is bridged by WGA (a, b) as opposed to the looser contact when attachment is mediated by ConA (c, d). a, c, X 16900; b, d, X20800.
The concentration of WGA at which maximal erythrocyte-macrophage interaction is established is remarkably lower than that required for reaching an optimum in the ConA-mediated interaction. At 37°C
2 pg WGA/ml suffice to establish the same number of stable cell-cell associations as those brought about by 5, 10 and 40 ,ug/ml (fig. 4A). The temperature at which cell-cell as-
Exp CellRes 103(1976)
Diffeerential lectin effects in erythrophagocytosis sociation is brought about is much less critical in the case of WGA-mediated interaction than with ConA-mediated association. At 2 pg WGA/ml there is a linear dependence of cell-cell interaction with temperature (fig. 4A). At 5, 10 and 40 pg WGA/ml6688 % of the interaction at 37°C was already observed at 8°C. A significant proportion of ingestion of attached erythrocytes was observed only at 30°C and 37°C and the higher the concentration of WGA in the macrophage treatment, the higher was the percent of ingested erythrocytes (fig. 4B, C). Macrophage interaction with ConA and WGA results in cross-linking of surface glycoproteins and cluster formation [7]. Lectin-macrophage interaction as reflected in both the amount of bound lectin, and its subsequent internalization is temperature dependent. The effect of temperature at which lectins were bound to macrophages on the subsequent attachment of erythrocytes, at different temperatures, was therefore studied. Fig. 5 shows the effect of exposure of macrophage monolayers to ConA at different temperatures on a subsequent erythrocyte attachment at 14, 18.5, 24 and 37°C. The highest degree of erythrocyte attachment obtained at any of the temperatures applied was that following a ConA treatment stage at 14 or 18S”C. The highest sensitivity to the temperature of lectin treatment was observed at a temperature of attachment of 14°C. Allowing for extensive cluster formation of ConA-glycoprotein conjugates at 24°C and 37°C reduces the chances for a subsequent erythrocyte attachment at 14°C to 10% and 2 % of maximal attachment, respectively. When cell-cell interaction is brought about at 24°C and 37”C, erythrocyte attachment is only slightly lower than that observed at 14-18.5”C. The data in fig. 5 further indicate the high temperature
285
dependence of ConA-mediated attachment i.e. 3-5-fold higher at 37°C than at any of the other temperatures at which the association was assessed. An analogous set of experiments to that represented in fig. 5 with WGA-mediated erythrocyte attachment indicates non-significant differences in attachment at any of the temperatures subsequent to macrophage treatment with WGA at 8, 14, 18.5, 24 and 37°C. A trend for a maximal attachment following WGA binding at 18.5”C was observed. Erythrocyte interaction with WGAtreated macrophages at 37°C results in a 40% ingestion of the interacting erythrocytes within 60 min. This is true for WGA interaction with macrophages at 8. 24. 18.5 and 24°C. When macrophages were exposed to WGA at 37°C the subsequent interiorization of attached erythrocytes decreased to 10% (100 % being 590 erythrocytes attached at 37”C/lOO macrophages). The difference in the characteristics of ConA- and WGA-mediated erythrocyte attachment to macrophages was further assessed by attempts to form rosettes under conditions of mild rotation or rocking. Under conditions of rotatory motion in the suspension (80 rotations/min) essentially no stable contact between erythrocytes and ConA- orWGA-treated macrophages was established (30 min, 37°C). A rocking motion (17 complete cycles of upward and downward motionsimin) sufficed to reduce to nli and to 30% erythrocyte interaction with ConA- and WGA-treated macrophages. respectively. Of the 210 erythrocytes associated with 100 M-WGA, 97 were ingested within the 30 min of incubation. Control values of interaction under nondisturbed exposure of erythrocyte suspensions to lectin-treated macrophages are given in fig. 2 (30 min). At this point it should be emphasized that the wash condi-
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tions after rosette formation consist of two periods of rotatory shaking in PBS. Thus, all attached erythrocytes are those that establish cell-cell contact withstanding the shear force of the rotatory motion. Electron micrographs of ConA- and WGA-mediated erythrocyte attachment to macrophages are given in fig. 6. Erythrocyte membranes show very extended and extremely tight interaction with WGAtreated macrophages at a temperature of interaction as low as 19°C (fig. 6a, b). A distinctly looser interaction is observed in the ConA-mediated attachment even when the interaction is brought about at 30°C (fig. 6~). At 19°C except for rare occasions (fig. 6d) the gaps between erythrocytes and macrophages are so wide that no certainty exists regarding a real interaction. This is most probably due to the fact that small areas of contact develop and while sectioning the chance to observe these areas is scant. A very tight interaction is also observed between erythrocytes attached to WGA-treated macrophages. A small section of two adjacent erythrocytes can be observed in fig. 6a. While counting erythrocytes associated with M-WGA, erythrocyteerythrocyte associations at the periphery were often observed. Clusters of erythrocytes are rarely ingested and are often the cause of the observation that ingestion does not exceed 40-50 %. It was also established that agglutination of erythrocytes via WGA does not lend itself to ready resuspension, and single cell suspensions could therefore not be obtained. This is the reason that erythrocytes coated with WGA were not amenable to parallel studies to those performed with ConA-coated erythrocytes [4]. WGA-mediated erythrocyte attachment to macrophages at 22°C was 80-90% reversible upon interaction with N-acetyl-nglucosamine (0.1 M, 30 min, 22°C) up to 3 h Exp Cell Res 103 (1976)
of interaction. At 6 h interaction at 22°C or 24 h at 19” only 70% and 35 %, respectively, could be detached by treatment with the inhibitor . During the enumeration of hundreds of macrophage-erythrocyte rosettes generated under the different conditions specified in the figures it became clear that ConA- and WGA-mediated cell-cell interaction differs in yet another aspect. While in the ConAmediated erythrocyte-macrophage interactions the erythrocytes were always localized at the very distant periphery of the cell, those attached via WGA assumed various locations, from peripheral at low temperatures or short interaction times to perinuclear at longer times of incubation at temperatures of 324°C. It was also notable that all the ingested erythrocytes assumed a distinct perinuclear position. In order to follow possible changes in erythrocyte location on the macrophage subsequent to its primary attachment, preformed rosettes (14”C, 30 min) were observed under phase contrast microscopy in a Nikon M microscope equipped with an incubator, set at 37°C. Single cells were followed for 30-60 min from the time of the temperature shift to 37°C and photographed at 3 min intervals. Fig. 7 represents 3 time points out of a sequence of pictures that were taken for (i) ConA-mediated and (ii) WGA-mediated rosette formation. The Fig. 7. Rel. movement of erythrocytes and yeast cells attached to macrophages via lectins. Macrophages were treated with either 20 pg/ml (a-c, g-i) ConA; or (d-f) WGA at 14”Cfor 30 min. Subsequent to x2 wash in PBS macrophage monolayers were incubated with 10’ (u-f) erythrocytes or (g<) yeast cells for 30 min at 14°C. Preformed rosettes were immediately washed in PBS. transferred to fresh PBS and incubated at 37°C for continuous microscopic observation. The time sequence represented in the figure is, macrophage-ConA-erythrocyte: (a) 10 min; (b) 20 min; (c) 25 min; macrophage-WGA-erythrocytes: (d) 10 min; (e) 23 min; v) 30 min; macrophage-Con&yeast cells: (g) 5 min; (h) 11 min; (i) 21 min. X 1320.
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lectin effects in erythrophagocytosis
2
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Table 1. SBA-mediated Macrophage treatment Neuraminidase * + + + + + + +
SBAC (pdml) J 5 40 40 -
erythrocyte
macrophagea
interaction
Erythrocyte treatment Neuraminidaseb
SBAC Q4h-4
+ + + + + +
2 2
No. attached erythrocytesllO0 macrophages 0 22 3 3 20 35 104 158 188
a Erythrocyte suspensions (7.5X lo6 cells/ml) were incubated with macrophages for 60 min at 22°C. b Neuraminidase treatment as described in Methods. c SBA treatment of macrophages and erythrocytes, 30 min, 22°C. Erythrocyte suspensions 75x lo6 cells/ml.
two erythrocytes seen in fig. 7a-c are attached at the distant periphery of the macrophage membrane. No changes in the spatial relation of the erythrocytes to the peripheral regions or to the nucleus were observed during the 25 min incubation at 37°C. At the same time, phase lucent vesicles derived from ConA pinosome fusion were generated [7, 111. Fig. 7d-f represents the sequence of erythrocyte movement subsequent to attachment to a WGAtreated macrophage. At 10 min, two erythrocytes were still located at a peripheral position, three of the other erythrocytes had a different plane of focus. At 23 min incubation at 37°C the two erythrocytes changed their position relatively to both the border of the cell and the nucleus. At 30 min four erythrocytes had a common plane of focus and the distances between them had decreased. From the above it is impossible to decide whether the erythrocytes move or whether the macrophage spreads over their surface in the undulating movement of the membrane and the whole segment of the interacting membrane with the attached erythrocyte assumes a more central position relative to the nucleus. Exp Cell Res 103 (1976)
The interaction of M-ConA with yeast cells has been characterised in some detail [5]. In the case of ConA-mediated attachment of yeast cells the attachment leads to yeast interiorization in contrast to erythrocyte behaviour when attached via ConA but conforming with erythrocyte ingestion when attachment is mediated by WGA. In order to assess whether the centripetal movement is a prerequisite for phagocytosis and whether ConA-treated macrophages are not distinct in this respect from WGAtreated macrophages, the dynamics of interaction of yeast cells with ConA-treated macrophages was followed. Fig. 7 (g-i) represents the interaction of preformed yeast cell macrophage rosettes (30 min, 14°C) at 5, 11 and 21 min subsequent to transfer to 37°C. The yeast cells can readily be seen to change their position relative to the periphery, nucleus and to each other. Under these conditions 70% of attached yeast cells are ingested during a 60 min incubation [S]. Erythrocytes coated with SBA, though readily agglutinable, failed to establish stable rosettes with macrophages. Recently it has been observed that SBA interaction with macrophages requires a first step of
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Table 2. A rough quantitative estimate of random surface distribution of ConA and VGA
Cell type
(pm?)
Ref.
No. bound ConA moleculeslcell (X 1OP) Ref.
Macrophage Erythrocyte Yeast ceil
2 500”
[24]
5
Surface area
50 60
[71
1 [51
3.4
Surface density (ConA/pm”)
No. bound WGA molecules/cell (X 10-y Ref.
2 wo
15”
20 000 57 000
3’ N.D.
E71
Surface density CNGAl~nPi 6 ooo 50000 N.D.
’ A minimum value [24] that might need a reconsideration in view of the many ruffles seen in scanning elecl;ron microscopy [23]. * A minimum value based on an estimate of 50% lectin interiorization. A three-fold value of WGA binding over ConA binding was obtained for SV40-transformed golden hamster cells [14]. ’ .4 three-fold value of ConA binding was taken as a first approximation.
neuraminidase treatment and that polymerized SBA binds also to non-treated macrophages [7]. The ability of SBA to mediate erythrocyte-macrophage interaction was therefore tested in combination with neuraminidase treatment of either one or both cell types. Table 1 indicates that a significant interaction via SBA requires a pretreatment of erythrocytes with neuraminidase. The mild vacuolation observed in macrophages treated with 40 pg/ml SBA implies that a certain fraction of the SBA was in an aggregated state [7]. When neuraminidase-treated erythrocytes were coated with SBA and incubated with macrophages the latter established stable association with the erythrocytes whether they were pretreated with neuraminidase or not. Transferring preformed rosettes to 37°C resulted in total dissociation of erythrocytes from macrophages. This is in line with a lower association constant of SBA to glycoproteins at 37°C than at 22°C. WBA-coated macrophages (30 pg/ml, 30 min. 22°C) interact with erythrocytes (10’ cellsiml, 60 min, 22°C) to yield rosettes consisting of .547&40 erythrocytes/lOO macrophages. Incubation of preformed rosettes at 37°C in medium for 90 min did not result in a detectable ingestion of attached erythro-
cytes. The number of attached erythrocytes was not reduced, however, during the additional incubation period. DISCUSSION The interaction of erythrocytes with macrophages coated with ConA or WGA results in the formation of stable cell-cell associations. “Stable” means in the present experimental design an association that withstands two steps of 20 set wash in PBS on a rotatory shaker operated at a speed of 100 rotations/min. At early phases of cell-cell contact the shear forces developing in the solution by the rotatory motion do not allow for stable associations, i.e. when the interacting cells are incubated together under rotatory shaking conditions rosette formation is precluded. Rosette formation strong ly depends on the concentration of ery-throcyte suspensions. At 5 x 10’ erythrocytes! ml, ConA-mediated rosette formation is far from saturation, reaching a value of 550 erythrocytes/ 100 macrophages. The interaction via WGA is more efficient, resulting in the association of about 1300 erytbrocytes/IOO macrophages (fig. 1). The time dependence of rosette formation at 37°C (fig. 2) further establishes the
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difference in rates at which the interaction via ConA and WGA approaches saturation. Since the number of collisions of erythrocytes and the macrophage monolayer is the same whether macrophages were coated with ConA or WGA the different rate must reflect a differential development of strength of binding. Moreover, the WGAmediated erythrocyte attachment to macrophages can, after a certain lag period, trigger erythrophagocytosis (fig. 2B). Thus the more efficient binding is followed by a subsequent step of phagocytosis. The lag period between attachment and ingestion could reflect the need for a circumferential attachment of the plasma membrane around the erythrocyte. If one regards the process of phagocytosis as the spreading of the macrophage plasma membrane around a particle analogously to its spreading on glass [13] then it is reasonable that the rate of spreading would reflect the affinity between the two surfaces, that of the macrophage and that of the particle. Most interesting is the observation that ConA-coated macrophages fail to complete the encircling of the attached erythrocyte while when coated with WGA they do so with a good efficacy; i.e. 40% of the attached erythrocytes are phagocytosed within 1 h. WBA and SBA are also capable of serving as a bridge for rosette formation, however, neither triggers erythrophagocytosis. For SBA mediates rosette formation it is essential that the erythrocytes be pretreated with neuraminidase. Macrophage pretreatment with neuraminidase is less critical for rosette formation though it increases the interaction (table 2). ConA-mediated erythrocyte attachment (hemadsorption) has a non-linear dependence on temperature with a 3-fold increase in the range of 1837°C. This is true at three different degrees of saturation of ConAExp Cell Res 103 11976)
binding sites on the macrophages. A similar pattern has been observed for the ConAmediated agglutination of suspended SV40transformed hamster cells [lo, Ill. In contrast Rittenhouse & Fox [ 151,studying hemadsorption to ConA-coated LM cells, observed a very sharp temperature effect, resembling a phase transition in the range of 12-18°C below which hemadsorption amounted to about 10% of that observed at 18°C and above which no further increase in hemadsorption has been observed. The sharp transition range could be shifted to a lower and a higher temperature range depending on the composition of fatty acids in membrane phospholipids [16]. ConAmediated yeast cell-macrophage association [5] has a less pronounced temperature dependence; 60-70% of the yeast cells attached to macrophages at 25°C were already attached at 15°C and no increase in attachment was observed in the range of 25-37°C. The difference in the temperature dependence of ConA-mediated attachment of erythrocytes and yeast cells brings up the contribution of ConA-binding sites on the counter cell to the interaction pattern. WGA-mediated hemadsorption is much less temperature dependent than that of the ConA-mediated hemadsorption (fig. 4). Macrophages precoated with 5, 10 and 40 pg WGA/ml interact to a similar degree with erythrocytes at 8-37°C. Precoating of macrophages with 2 pg WGA/ml results in sensitized cells with a hemadsorption capacity that is extremely temperature sensitive. It is possible that cell-bound WGA is distributed very sparsely on the membrane and lectin receptor conjugates or small crosslinked clusters have to be aggregated at the area of apposition of two membranes for development of stable contacts. The lateral mobility of membrane glycoproteins increases with temperature [ 17, 181.It is note-
Differential lectin effects in erythrophagocytasis worthy that at 37°C hemadsorption to macrophages coated with a solution of 2 pg WGAlml reaches the level of maximal hemadsorption achieved under conditions of higher saturation of lectin-binding sites on the macrophage membrane (fig. 4). The ingestion efficiency of macrophages for attached erythrocytes does, however, decrease in rosettes formed with macrophages of a lower degree of saturation in WGAbinding sites (fig. 4B, C). In comparing the degree of hemadsorption achieved by precoating macrophages with WGA and ConA at various concentrations it is important to take into account the different physical parameters of both lectins. ConA and WGA differ markedly in their molecular dimensions (102000 and 36000 D mol. wt, respectively [19-201) though both possess four sugar-binding sites. Macrophage-binding sites for WGA are more abundant than those for ConA as was also observed for other cell types [6, 7, lo]. It is interesting that Vlodavsky & Sachs [lo] report a higher apparent association constant for membrane glycoproteins for ConA (lo7 M-l) than for WGA (1.6~ IO6M-l). The striking difference between the pattern of rosette formation and the fate of attached erythrocytes is fully expressed even when the compared conditions consist of 5 pg/ml of WGA and 40 pg/ml of ConA in the coating so&ion. ConA-mediated cluster formation of glycoproteins requires a short range lateral movement of the latter compounds. Once they are clustered and cross-linked by ConA their lateral diffusion is much hindered [I7]. Both processes, initial cluster formation and redistribution of cluster components are temperature dependent, It is only plausible that the redistribution of glycoprotein-receptor conjugates will be
291
more sensitive to temperature than that of the non-cross-linked glycoproteins. Pectin coating of macrophages at different temperatures results in various degrees of cluster formation as well as in interiorization of lectin receptor conjugates [7]. When the interaction of ConA with macrophages is carried out at 2537°C the clustering is such that a shift to 14°C for a period of incubation with erythrocytes does not allow for extensive redistribution of receptors and extensive hemadsorption. If incubation with erythrocytes is carried out at 24°C or better still, at 37”C, redistribution of clusters allows for hemadsorption amounting to 70% of maximal observed hemadsorption, The highest value of hemadsorption is obtained following a lectin-coating step carried out at 14-18°C. The physical state of membrane phospholipids appears to be highly sensitive to variations in this temperature range [ 181. Hemadsorption mediated by WGA is much less influenced by the temperature at which macrophages are pretreated with the lectin. The characteristic differences in ConAand WGA-mediated hemadsorption are in line with those observed for lectin-mediated agglutination of SV40-transformed hamster cells [lo, 141and for agglutination of erythrocytes [S, 91. Mechanical shear introduced by shaking interferes strongly with ConAmediated erythrocyte agglutination, but has only a small effect on WGA-mediated agglutination [$I. The data reported above indicates a higher resistance to shear forces of WGA-mediated erythrocyte-macrophage rosettes than that of ConA-mediated rosettes. Sachs et al. [lo, 14, 211 propose that ConA-binding sites have a high short range lateral mobility that has to be restricted before effective agglutination or ceil binding to ConA-coated nylon fibres takes place.
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Goldman and Bursuker
According to this criteria the more abundant WGA-binding sites have a much lower lateral mobility and therefore WGAmediated agglutination or attachment to fibres is much less temperature sensitive [IO, 14,211. ConA- and WGA-binding sites may reside on different glycoproteins or at least the overlap in receptors for either lectin may involve a small fraction of the binding entities [6]. In contrast to the manifold data regarding the factors governing the surface distribution of ConA-binding sites the data about the lateral mobility of WGA-binding sites is rather scant. On the basis of the above discussed facts, inferences and a rough estimate of the quantitative parameters involved (table 2), the following scheme is suggested for the differential effect of ConA and WGA on erythrocyte attachment and ingestion. Phagocytosis requires a multibridge interaction along the entire circumference of the particle. When lectin molecules bound to macrophages serve as a bridge the primary attachment area as well as the membrane enveloping the particle have to contain lectin receptor conjugates. Under conditions of extensive clustering of lectin receptor conjugates as those implied to develop in ConA-treated macrophages the plasma membrane surrounding the area of erythrocyte attachment may be deprived of, or poor in, free lectin-binding sites and as a result no stable circumferential interaction can be established. Since not all of the ConA-binding sites are saturated under the experimental conditions, addition of ConA to preformed rosettes that do not undergo erythrocyte ingestion triggers full erythrophagocytosis of attached erythrocytes [4]. The additional ConA may coat both the macrophages as well as the erythrocytes. WGA-binding sites are more abundant on Exp Cell Res 103 (1976)
the macrophage than ConA-binding sites. If in addition they exhibit lower tendency for cluster formation [IO, 14, 211 then WGA distribution on the macrophage plasma membrane may enable both the interaction with erythrocytes leading to attachment and the subsequent interaction of adjacent plasma membrane to achieve circumferential interaction. A very tight interaction and membrane “crawling” to envelop erythrocytes attached to macrophages via WGA is indicated in fig. 6. It should be kept in mind that due to the high mobility of surface receptors many receptors can be aggregated at interaction sites with the particle. Taking a value of 1.5~ low9 cm2/sec for the diffusion constant of surface macromolecules [17] then a glycoprotein molecule can cover a distance of 6 ,u/min. Edidin & Fambrough [17] have shown that cross-linking of surface macromolecules labelled with Fab by an antiFab immunoglobulin reduces the rate of surface spread of Fab. They have estimated a reduction by a factor of 10 (0.15~ lops cm2/ set) in the diffusion constant. In analogy therefore it is expected that cross-linking of glycoprotein receptors via ConA will reduce their mobility by a factor of -3 (X2= 4 Dt). The estimated mobility of a surface cross-linked macromolecule is still very high relative to cellular distances, and cluster formation should therefore be regarded as a dynamic process; in a time scale of minutes small clusters should readily change place and reorganize unless crosslinked to an external particle (yeast, erythrocyte). Big clusters (patches) induced by polyvalent lectins may have a different, larger, time factor for spreading and reorganization. Erythrocytes tend to establish the primary contact with the macrophage at cell peripheral regions where the undulating membrane is flat and well spread out on the
Differential lectin effects in erythrophagoqvtosk
293
glass. This is true for both ConA- and teriorization is clearly suggested from the WGA-mediated attachment. When attach- differential behaviour of ConA-mediated ment is not followed by an ingestion step yeast cell and erythrocyte-macrophage ro(ConA-mediated erythrocyte-macrophage settes. The density of ConA-binding sites rosettes) the erythrocytes do not change on mouse erythrocytes is about 3-fold lower than that observed for yeast ce!ls (table 2) their position during prolonged incubation periods. Attachment that is followed by an where a closely packed monolayer of ConA ingestion step (WGA-mediated macro- is accommodated at ConA saturation ]:5]. phage-erythrocyte rosettes and ConA- Thus, under comparable conditions of macmediated macrophage-yeast cell rosettes) rophages coating with ConA, the yeast cell results in a subsequent movement of the is more efficient than erythrocytes in reattached cells to a perinuclear position and cruiting ConA receptor conjugates and in only at this position phagocytosis seems to establishing stable circumferential intertake place. It is impossible to decide at this actions. As a result, yeast cells are ingested stage whether the erythrocyte moves or with very high efficiency while the erythrowhether it is encompassed by the plasma cytes are not. It is interesting to note that membrane at distal edges and then the yeast cells attached to macrophages whole complex of undulating membrane en- changed their position on the macrophage veloping the erythrocyte moves towards with time much the same as erythrocytes the centre. The latter possibility is more attached via WGA (compare fig. 7g-i with plausible. Membrane envelopment of 7d-j). erythrocyte at distal regions of the macrophages prior to phagocytosis have been obREFERENCES served using scanning electron microscopy 1. Mantovani, B, Rabinovitch. M & Nussenzweig, Z, [22]. Another possible mechanism that was J exp med 135 (1972) 780. 2. Griffin, F M. Jr & Silverstein, S C. J exp med 139 considered is the formation of craters (1974) 323. through which particles are ingested. Kap3. Griffin, F M, Jr, Griffin, J A, Letder, J E & Silverstein, S C, J exp med 142(1975) I263. lan et al. [22] suggest that this mechanism is 4. Goldman, R & Cooper. R A. Exp celires 95 (1975) restricted to small phagocytosible particles 223. 5. Bar-Shavit, Z & Goldman, R. Exp csii res 99 and that this phagocytosis is mostly peri(1976) 221. nuclear. Polliack & Gordon [23] have ob- 6. Nicolson, G L, Int rev cytol 39 (1974) 89. 7. Goldman, R, Sharon. N & Lotan, R, Exp cei! res served crater formation during the inter99 (1976) 408. action of macrophages with erythrocytes. 8. Schnebli, H P & Bachi. T, Exp cell res ?! (1975) 175. Accepting either model it is clear that 9. Vlodavsky, I, Inbar, M & Sachs, L, Biochim biotriggering the steps following attachment be phys acta 274 (1972) 364. it membrane crawling around the particle or IO. Vlodavsky, I & Sachs. L, Exp cell res 9; (1975) 202. the formation of craters due to sinking-in of II. Goldman, R & Raz, A, Exp cell res 96 (1975) 393. 12. Spurr, A R, J ultrascruct res 26 (1969) 3 1. the membrane would depend on additional Stossel. T P, Seminars in hematol 12 (1975) 83. interaction between the particle and the 13. 14. Vlodavsky, I & Sachs. L. Exp cell res 93 (1975) 111. plasma membrane. These events would be H G & Fox, C F, Biochem biophys both temperature and time dependent (figs 1.5. Rittenhouse, i-es commun 57 ( 1974)323, 16. Rittenhouse. H G. Williams. R E & Fox, C F, J 2,4: 7). supramol struct 2 (1974) 629. The importance of the characteristics of 17. Edidin, M & Fambrough, D, J ceh bio? 57 (1973) 27. the ingested particles in triggering the in-
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18. Petit, V A & Edidin, M, Science 184 (1974) 1183. 19. Reeke, G N, Becker, J W, Cunningham, B A, Gunther, G R, Wang, J L & Edelman, G M, Ann NY acad sci 234 (1974) 369. 20. Nagata, Y &Burger, M M, J biol them 249 (1974) 3116. 21. Rutishauser, U & Sachs, L, J cell biol66 (1975) 76.
15x-pCell Res 103 (1976)
22. Kaplan, G, Gandernack, G & Seljelid, R, Exp cell res 95 (1975) 365. 23. Polliack, A&Gordon, S, Lab invest 33 (1975)469. 24. Werb, Z &Cohn, Z A, J exp med 134(1971) 1570. Received June 9, 1976 Accepted June 11, 1976