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Surface membrane changes in mitogen-transformed lymphocytes
Preliminary notes
43 1
action of colchicine to reduce the selective The expression of new surface markers durenzyme release and explained the DzO ef- ing mitosis has been shown using cultured fect from its property to favor the assembly mouse leukaemia cells [l]. Other workers of microtubules. have reported mitogen-induced surface Apparently phagocytic metabolism in changes in rat thymocytes [2] and mouse leucocytes is controlled by a similar mech- lymphocytes [3,4]. We have previously reanism as that which exerts secretion. DzO, ported that fluorescent and peroxidase conConA [6] and calcium ions [7] which are jugates of the non-mitogenic lectin wheatknown to favor the assembly of micro- germ agglutinin @VGA) normally label only tubules, stimulate the metabolism and myeloid cells, such as polymorphonuclears CAMP which is antagonistic to these com- and macrophages, from mouse spleen and pounds [4], inhibits it. Induction of phago- other lymphoid tissues [5, 61. It was noted, cytic metabolism [8] might involve the as- however, that lymph nodes from infected sembly of microtubule system which con- mice contained unusually high numbers of currently stimulates the release of lyso- cells which were positive for WGA. This efsomal enzymes. fect could be simulated in vitro by culturing lymph node cells from healthy mice with References concanavalin A (ConA) or phytohaemag1. Romeo, D, Jug, M, Zabucchi, G & Rossi, F, FEBS glutinin (PHA), lectins which are mitogenic lett 42 (1974) 90. 2. Romeo, D, Zabucchi, G, Miani, N & Rossi, F, for T lymphocytes. Previous work has esNature 253 (1975) 542. tablished that the labelled cells in lymph 3. Nakagawara, A, Takeshige, K & Minakami, S, Exp nodes have the morphological characcell res 87 (1974) 392. 4. Gillespie, E, Ann NY acad sci 253 (1975) 771. teristics of transformed lymphocytes [6]. 5. Zurier, R B, Weissmann, G & Hoffstein, S, J clin In this study the stage in transformation invest 53 (1974) 297. 6. Hoffstein, S, Soberman, R, Goldstein, I & Weiss- at which these surface changes occur has mann, G, J cell bio168 (1976) 781. 7. Gautvik, K M & Tashjian, Jr A H, Endocrinol 93 been investigated by correlating the ap(1973) 793. pearance of WGA-labelled cells with DNA 8. Sbarra, A J & Kamovsky, M L, J biol them 234 synthesis in whole cultures. We have cul(1959) 1355. tured lymphocytes in the presence of bacReceived May 13, 1976 terial lipopolysaccharide to determine Accepted July 19, 1976 whether B cells stimulated under these conditions also express this surface marker. A double-labelling technique has been used Surface membrane changes in mitogento see whether cells carrying WGA markers transformed lymphocytes also bear surface components characteristic P. J. ROBINSON,’ B. H. ANDERTON* and I. M. of B or T lymphocytes, both in stimulated ROITT,’ ‘Department of immunology, Middlesex Hospital Medical School, London, WlP 9PG, and lymphocyte cultures and in a number of ?School of Engineeting and Science, Polytechnic of lymphocyte-derived tumour cell lines. Central London, London, WIM 8JS, UK .
I
Summarv. Resting lvmuhocvtes from the lymph nodes of normal mice are labelled only weakly by fluorescent conjugates of the lectin wheatgerm agglutinin (WGA). In contrast, lymphocytes cultured in the presence of B or T cell mitogens are strongly labelled. The onset of this change is-shown to OCI% prior to the uptake of [aH]TdR, suggesting that this surface change occurs before cell division.
Materials and Methods Lectins and mitogens. Wheatgerm agglutinin (WGA) was prepared by the method of Allen et al. [7]. ConA, type IV, was purchased from Sigma London Chemical Co. Phytohaemagglutinin (PHA) was obtained from Wellcome Laboratories, Beckenham, UK, and S. Marcescens lipopolysaccharide W from Difco Ltd. Exp CeNRes 102(1976)
432
Preliminary notes paper and the radioactive content of each sample measured in a liquid scintillation spectrometer. Culture of turnours. Ll210 cells were grown in CBA xDBA/Z mice. 5 x IO6 cells were injected intraperitoneally and cultures harvested 7-10 days later. EL4 lymphoma cells were cultured similarly but in CS7 BL mice. MOPC 104E cells were grown in BALB/c mice by subcutaneous injections of 10’ cells. The resulting solid tumours were removed two weeks later and the cells teased into fresh medium.
Results
20406080
20 40
60
80
Fig. i. Abscissa: time (hours) of culture; ordinafcs: (left) % of cells WGA positive (0-O); (righ?) TdR incorp. (cpm/h) X 10e3(0-- -0). Bars S.D. WGA labelling of and DNA synthesis by mouse lymph node cells cultured in the presence of (a) PHA; (b) ConA; (c) LPS; (d) control. Lymphocytes were prepared from the pooled lymph nodes of BALB/c mice and, after washing, resuspended at a concentration of 6~10~ cells/ml in RPM1 1640medium containing 10% fetal bovine serum with added glutamine (2 mM), benzylpenicillin and streptomycin. PHA or ConA (1 pg/rnl) or lipopolysaccharide (LPS) (25 pg/ml) were added in volumes insufficient to cause a significant change in the cell concentration. Cultures without added mitogens acted as controls. Transformation was carried out in % well Cooke microtiter plates using 1.2~ 10” cells/well (200 ~1). Samples were taken at various times; the cells were washed twice with cold phosphate-buffered saline (PBS) and resuspended in 50 ~1 of cold PBS containing 25 pg/ml of fluorescein-conjugated WGA, aggregates being disrupted by gently passing the suspension through a fine needle with a syringe. The proportion of stained cells was determined immediately using a Leitz Ortholux ultraviolet microscope with incident illumination. Only cells with strong clear ring staining were counted as WGA-positive while cells with weak or hazy uniform fluorescence were scored as negative. The degree of lymphocyte proliferation was measured by assaying the rate of incorporation of 13H1TdR into the DNA of cells in the remaining wells; at selected times 1 &i of carrier-free methylJ3H]TdR in 50 ~1 of RPMI+ 10% fetal bovine serum was added to each well and culture continued for a further hour. The plates were frozen and stored at -20°C until cultures were harvested using a semi-automatic cell harvester. TCA-precipitable material was collected on glass fibre Exp CellRes 102(1976)
Effects of transformation upon WGA binding sites. Fig. 1 shows the proportion of cells stained with WGA at various times over a complete transformation period and this is compared to the rate of DNA synthesis in parallel cultures. Study of the timecourse of the response in PHA and ConAstimulated cultures shows that an increase in the number of WGA labelled cells is first detectable after 15h, whereas an increase in the rate of DNA synthesis becomes appreciable only after 25 h (fig. la, b). The proportion of WGA-positive cells in PHA and ConA-stimulated cultures is maximal at around 40 h, although the percentage of blast cells continues to increase up to 85 h. Consistent with this we find large cells at all stages of culture which do not label with WGA, the proportion apparently increasing with time. Transformation with LPS proceeds at a slower rate than with PHA or ConA and thymidine incorporation and the proportion of WGA-positive cells continues to increase throughout the transformation period (fig. 1c, d). To confirm that the number of WGApositive cells increases before thymidine incorporation becomes appreciable, we have investigated the period in which these changes occur in greater detail (fig. 2). The results show that lymphocytes stimulated with T-cell mitogens express binding sites for WGA early in the transformation period and that a plateau is
Preliminary
16o 3Or b
notes
433
the anti-brain serum and 3-4 % of the WGApositive cells carried surface immunoglobulin. In cultures stimulated with LPS, all WGA-positive cells carried surface immunoglobulin and none stained for both WGA and theta markers. In control cultures none of the few immunoglobulin-staining cells were WGA positive and we conclude that in stimulated cultures, WGA-labelled cells are unlikely to be myeloid cells carrying cytophilic immunoglobulin. Surface markers on tumour cell lines. All Ii’ cells taken from the peritoneal cavities of t mice carrying the Ll210 and the EL4 lymphomas were strongly labelled with the WGA conjugate. Cells taken from the 20 25 30 35 40 MOPC 104E tumour were not labelled Fig. 2. Abscissa: time (hours) of culture; orcli~~te.s: under these conditions, and it was imposas for fk. 1. Bars S.D. WGA-labelling of and DNA synthesis by mouse sible to label or agglutinate these cells with lymph node cells cultured in the presence of (a) PHA; WGA even at high concentrations. (b) ConA; (c) control. L1210 cells were found to be positive for reached before the peak of DNA synthesis. immunoglobulin but were not labelled by In this experiment the control levels of the anti-brain serum, whilst EL4 cells were WGA-positive cells were higher than those positive with the anti-brain serum but did of previous experiments, but the proportion not carry surface immunoglobulin. of stained cells in PHA- and ConA-stimulated cultures was correspondingly greater. Discussion Identi’cation of other surface markers on A number of authors have reported the aptransformed lymphocytes. To establish the pearance of new surface markers on lymlineage of the blast cells present after 40 h phocytes during transformation. Little is of culture, we used a double-labelling tech- known, however, of the nature of these nique similar to that previously described components or of their relationship with the [5]. Immunoglobulin- and theta-positive functional state of the cell. Boyd et al. and cells were identified using rabbit anti-mouse Schirrmacher & Festenstein have described immunoglobulin serum or appropriately ab- a B lymphocyte antigen which is immunosorbed rabbit anti-mouse brain serum [8], logically related to a component expressed respectively, followed by a goat anti-rabbit on transformed T lymphocytes. Both immunoglobulin antibody conjugated with groups suggest a convergence of the B and rhodamine. Thus it was possible to deter- T lymphocyte differentiation pathways mine if cells labelled with fluorescein-con- after leaving the ‘resting’ stage. Other jugated WGA also carried the surface authors have shown that activated lymphomarkers of B or T lymphocytes. In cultures cytes express a number of properties, such stimulated with PHA or ConA, 95 % of the as endocytosis of particulate matter [9] and cells positive for WGA also labelled with abundant receptors for immunoglobulin [ 101 160
Exp Cell Res 102 (1976)
434
Preliminary
notes
normally associated with myeloid cells. Although structural identity has not yet been established between the lymphocyte and myelocyte WGA markers, our previous results combined with those presented here might suggest a convergence not only of B and T cell pathways, but of the myeloid pathway also. The appearance of WGA-binding sites on the lymphocyte membrane is associated with the end of the ‘resting’ stage as the cell prepares to divide, and this raises the possibility that this surface component may be related to mitosis. The two lymphomas studied, one putatively B cell-derived and the other T, showed positive binding of WGA throughout the division cycle, whereas the plasma cell tumour was invariably negative. This recalls the absence of WGA sites on plasmocytes from normal spleens and suggests that WGA sites are not a general characteristic of all dividing cells. However, anomalous behaviour of cell surface components on neoplastic cells cannot be excluded, and further work is required to establish whether there is any general relationship of WGA sites to differentiation or mitosis.
9. McIntyre, J A, La Via, M M, Prater, T F K & Niblack, G D, Lab invest 29 (1973) 703. 10. Yoshida, T 0 & Andersson, B, Stand j immunol 1 (1972) 401. Received May 18, 1976 Accepted July 19, 1976
A serum protein that stimulates macrophage movement, chemotaxis and spreading E. J. LEONARD and ALISON SKEEL, Zmmunopathology Section, Laboratory of Immunobiology, National Cancer Institute, Bethesda, MD 20015, USA Summary. A normal serum protein, purified by gel filtration and ion exchange chromatography, enhanced the chemotactic response of mouse peritoneal macrophages to EAMS (endotoxin-activated mouse serum) and increased the number of migrated macrophages in the absence of EAMS. The protein also enhanced macrophage spreading. The Sephadex G-200 distribution coefficient indicated a molecular weight of about 100000 D.
The response of mononuclear phagocytes to chemotactic factors has been studied in chambers divided into upper and lower compartments by a membrane through which cells can pass [l-4]. Cells in the upper chamber respond to chemotactic stimuli in the lower chamber by migrating through pores in the membrane and spreading on its under surface. The magnitude of This work was supported by the MRC of Great Britain. the response is quantified by a count of the number of cells/unit area of membrane. We found that mouse peritoneal macrophages References did not respond to chemotactic stimuli in 1. Thomas, DB, Nature 233 (1971) 317. 2. Lamont, J T, Perotto, J L, Weiser, M M & Is- the lower chamber unless serum was added selbacher, K J, Proc natl acad sci US 71 (1974) to the tissue culture medium in which the 3726. 3. Boyd, R L, Rolland, J M & Couchi, M N, Imm cells were suspended. We now show that a comm 3 (1974) 337. single serum protein is responsible for this 4. Schimnacher, V & Festenstein, H, J imeffect. Furthermore, the action of the promunogenetics 2 (1975) 337. 5. Robinson,PJ&Roitt,IM,Nature250(1974)517. tein is reflected not only in chemotactic 6. Robinson, P J, Penfold, P L & Roitt, I M, Clin exp responses, but in enhanced macrophage immuno123 (1976) 98. 7. Allen, A K, Neuberger, A & Sharon, N, Biochem j movement in the absence of a chemotactic 131(1973) 155. 8. Gybngybssy, M I C & Playfair, J H L, Cell im- stimulus and in marked spreading on Petri munol7 (1973) 118. dish surfaces. Exp Cell Res 102 (1976)
43 1
action of colchicine to reduce the selective The expression of new surface markers durenzyme release and explained the DzO ef- ing mitosis has been shown using cultured fect from its property to favor the assembly mouse leukaemia cells [l]. Other workers of microtubules. have reported mitogen-induced surface Apparently phagocytic metabolism in changes in rat thymocytes [2] and mouse leucocytes is controlled by a similar mech- lymphocytes [3,4]. We have previously reanism as that which exerts secretion. DzO, ported that fluorescent and peroxidase conConA [6] and calcium ions [7] which are jugates of the non-mitogenic lectin wheatknown to favor the assembly of micro- germ agglutinin @VGA) normally label only tubules, stimulate the metabolism and myeloid cells, such as polymorphonuclears CAMP which is antagonistic to these com- and macrophages, from mouse spleen and pounds [4], inhibits it. Induction of phago- other lymphoid tissues [5, 61. It was noted, cytic metabolism [8] might involve the as- however, that lymph nodes from infected sembly of microtubule system which con- mice contained unusually high numbers of currently stimulates the release of lyso- cells which were positive for WGA. This efsomal enzymes. fect could be simulated in vitro by culturing lymph node cells from healthy mice with References concanavalin A (ConA) or phytohaemag1. Romeo, D, Jug, M, Zabucchi, G & Rossi, F, FEBS glutinin (PHA), lectins which are mitogenic lett 42 (1974) 90. 2. Romeo, D, Zabucchi, G, Miani, N & Rossi, F, for T lymphocytes. Previous work has esNature 253 (1975) 542. tablished that the labelled cells in lymph 3. Nakagawara, A, Takeshige, K & Minakami, S, Exp nodes have the morphological characcell res 87 (1974) 392. 4. Gillespie, E, Ann NY acad sci 253 (1975) 771. teristics of transformed lymphocytes [6]. 5. Zurier, R B, Weissmann, G & Hoffstein, S, J clin In this study the stage in transformation invest 53 (1974) 297. 6. Hoffstein, S, Soberman, R, Goldstein, I & Weiss- at which these surface changes occur has mann, G, J cell bio168 (1976) 781. 7. Gautvik, K M & Tashjian, Jr A H, Endocrinol 93 been investigated by correlating the ap(1973) 793. pearance of WGA-labelled cells with DNA 8. Sbarra, A J & Kamovsky, M L, J biol them 234 synthesis in whole cultures. We have cul(1959) 1355. tured lymphocytes in the presence of bacReceived May 13, 1976 terial lipopolysaccharide to determine Accepted July 19, 1976 whether B cells stimulated under these conditions also express this surface marker. A double-labelling technique has been used Surface membrane changes in mitogento see whether cells carrying WGA markers transformed lymphocytes also bear surface components characteristic P. J. ROBINSON,’ B. H. ANDERTON* and I. M. of B or T lymphocytes, both in stimulated ROITT,’ ‘Department of immunology, Middlesex Hospital Medical School, London, WlP 9PG, and lymphocyte cultures and in a number of ?School of Engineeting and Science, Polytechnic of lymphocyte-derived tumour cell lines. Central London, London, WIM 8JS, UK .
I
Summarv. Resting lvmuhocvtes from the lymph nodes of normal mice are labelled only weakly by fluorescent conjugates of the lectin wheatgerm agglutinin (WGA). In contrast, lymphocytes cultured in the presence of B or T cell mitogens are strongly labelled. The onset of this change is-shown to OCI% prior to the uptake of [aH]TdR, suggesting that this surface change occurs before cell division.
Materials and Methods Lectins and mitogens. Wheatgerm agglutinin (WGA) was prepared by the method of Allen et al. [7]. ConA, type IV, was purchased from Sigma London Chemical Co. Phytohaemagglutinin (PHA) was obtained from Wellcome Laboratories, Beckenham, UK, and S. Marcescens lipopolysaccharide W from Difco Ltd. Exp CeNRes 102(1976)
432
Preliminary notes paper and the radioactive content of each sample measured in a liquid scintillation spectrometer. Culture of turnours. Ll210 cells were grown in CBA xDBA/Z mice. 5 x IO6 cells were injected intraperitoneally and cultures harvested 7-10 days later. EL4 lymphoma cells were cultured similarly but in CS7 BL mice. MOPC 104E cells were grown in BALB/c mice by subcutaneous injections of 10’ cells. The resulting solid tumours were removed two weeks later and the cells teased into fresh medium.
Results
20406080
20 40
60
80
Fig. i. Abscissa: time (hours) of culture; ordinafcs: (left) % of cells WGA positive (0-O); (righ?) TdR incorp. (cpm/h) X 10e3(0-- -0). Bars S.D. WGA labelling of and DNA synthesis by mouse lymph node cells cultured in the presence of (a) PHA; (b) ConA; (c) LPS; (d) control. Lymphocytes were prepared from the pooled lymph nodes of BALB/c mice and, after washing, resuspended at a concentration of 6~10~ cells/ml in RPM1 1640medium containing 10% fetal bovine serum with added glutamine (2 mM), benzylpenicillin and streptomycin. PHA or ConA (1 pg/rnl) or lipopolysaccharide (LPS) (25 pg/ml) were added in volumes insufficient to cause a significant change in the cell concentration. Cultures without added mitogens acted as controls. Transformation was carried out in % well Cooke microtiter plates using 1.2~ 10” cells/well (200 ~1). Samples were taken at various times; the cells were washed twice with cold phosphate-buffered saline (PBS) and resuspended in 50 ~1 of cold PBS containing 25 pg/ml of fluorescein-conjugated WGA, aggregates being disrupted by gently passing the suspension through a fine needle with a syringe. The proportion of stained cells was determined immediately using a Leitz Ortholux ultraviolet microscope with incident illumination. Only cells with strong clear ring staining were counted as WGA-positive while cells with weak or hazy uniform fluorescence were scored as negative. The degree of lymphocyte proliferation was measured by assaying the rate of incorporation of 13H1TdR into the DNA of cells in the remaining wells; at selected times 1 &i of carrier-free methylJ3H]TdR in 50 ~1 of RPMI+ 10% fetal bovine serum was added to each well and culture continued for a further hour. The plates were frozen and stored at -20°C until cultures were harvested using a semi-automatic cell harvester. TCA-precipitable material was collected on glass fibre Exp CellRes 102(1976)
Effects of transformation upon WGA binding sites. Fig. 1 shows the proportion of cells stained with WGA at various times over a complete transformation period and this is compared to the rate of DNA synthesis in parallel cultures. Study of the timecourse of the response in PHA and ConAstimulated cultures shows that an increase in the number of WGA labelled cells is first detectable after 15h, whereas an increase in the rate of DNA synthesis becomes appreciable only after 25 h (fig. la, b). The proportion of WGA-positive cells in PHA and ConA-stimulated cultures is maximal at around 40 h, although the percentage of blast cells continues to increase up to 85 h. Consistent with this we find large cells at all stages of culture which do not label with WGA, the proportion apparently increasing with time. Transformation with LPS proceeds at a slower rate than with PHA or ConA and thymidine incorporation and the proportion of WGA-positive cells continues to increase throughout the transformation period (fig. 1c, d). To confirm that the number of WGApositive cells increases before thymidine incorporation becomes appreciable, we have investigated the period in which these changes occur in greater detail (fig. 2). The results show that lymphocytes stimulated with T-cell mitogens express binding sites for WGA early in the transformation period and that a plateau is
Preliminary
16o 3Or b
notes
433
the anti-brain serum and 3-4 % of the WGApositive cells carried surface immunoglobulin. In cultures stimulated with LPS, all WGA-positive cells carried surface immunoglobulin and none stained for both WGA and theta markers. In control cultures none of the few immunoglobulin-staining cells were WGA positive and we conclude that in stimulated cultures, WGA-labelled cells are unlikely to be myeloid cells carrying cytophilic immunoglobulin. Surface markers on tumour cell lines. All Ii’ cells taken from the peritoneal cavities of t mice carrying the Ll210 and the EL4 lymphomas were strongly labelled with the WGA conjugate. Cells taken from the 20 25 30 35 40 MOPC 104E tumour were not labelled Fig. 2. Abscissa: time (hours) of culture; orcli~~te.s: under these conditions, and it was imposas for fk. 1. Bars S.D. WGA-labelling of and DNA synthesis by mouse sible to label or agglutinate these cells with lymph node cells cultured in the presence of (a) PHA; WGA even at high concentrations. (b) ConA; (c) control. L1210 cells were found to be positive for reached before the peak of DNA synthesis. immunoglobulin but were not labelled by In this experiment the control levels of the anti-brain serum, whilst EL4 cells were WGA-positive cells were higher than those positive with the anti-brain serum but did of previous experiments, but the proportion not carry surface immunoglobulin. of stained cells in PHA- and ConA-stimulated cultures was correspondingly greater. Discussion Identi’cation of other surface markers on A number of authors have reported the aptransformed lymphocytes. To establish the pearance of new surface markers on lymlineage of the blast cells present after 40 h phocytes during transformation. Little is of culture, we used a double-labelling tech- known, however, of the nature of these nique similar to that previously described components or of their relationship with the [5]. Immunoglobulin- and theta-positive functional state of the cell. Boyd et al. and cells were identified using rabbit anti-mouse Schirrmacher & Festenstein have described immunoglobulin serum or appropriately ab- a B lymphocyte antigen which is immunosorbed rabbit anti-mouse brain serum [8], logically related to a component expressed respectively, followed by a goat anti-rabbit on transformed T lymphocytes. Both immunoglobulin antibody conjugated with groups suggest a convergence of the B and rhodamine. Thus it was possible to deter- T lymphocyte differentiation pathways mine if cells labelled with fluorescein-con- after leaving the ‘resting’ stage. Other jugated WGA also carried the surface authors have shown that activated lymphomarkers of B or T lymphocytes. In cultures cytes express a number of properties, such stimulated with PHA or ConA, 95 % of the as endocytosis of particulate matter [9] and cells positive for WGA also labelled with abundant receptors for immunoglobulin [ 101 160
Exp Cell Res 102 (1976)
434
Preliminary
notes
normally associated with myeloid cells. Although structural identity has not yet been established between the lymphocyte and myelocyte WGA markers, our previous results combined with those presented here might suggest a convergence not only of B and T cell pathways, but of the myeloid pathway also. The appearance of WGA-binding sites on the lymphocyte membrane is associated with the end of the ‘resting’ stage as the cell prepares to divide, and this raises the possibility that this surface component may be related to mitosis. The two lymphomas studied, one putatively B cell-derived and the other T, showed positive binding of WGA throughout the division cycle, whereas the plasma cell tumour was invariably negative. This recalls the absence of WGA sites on plasmocytes from normal spleens and suggests that WGA sites are not a general characteristic of all dividing cells. However, anomalous behaviour of cell surface components on neoplastic cells cannot be excluded, and further work is required to establish whether there is any general relationship of WGA sites to differentiation or mitosis.
9. McIntyre, J A, La Via, M M, Prater, T F K & Niblack, G D, Lab invest 29 (1973) 703. 10. Yoshida, T 0 & Andersson, B, Stand j immunol 1 (1972) 401. Received May 18, 1976 Accepted July 19, 1976
A serum protein that stimulates macrophage movement, chemotaxis and spreading E. J. LEONARD and ALISON SKEEL, Zmmunopathology Section, Laboratory of Immunobiology, National Cancer Institute, Bethesda, MD 20015, USA Summary. A normal serum protein, purified by gel filtration and ion exchange chromatography, enhanced the chemotactic response of mouse peritoneal macrophages to EAMS (endotoxin-activated mouse serum) and increased the number of migrated macrophages in the absence of EAMS. The protein also enhanced macrophage spreading. The Sephadex G-200 distribution coefficient indicated a molecular weight of about 100000 D.
The response of mononuclear phagocytes to chemotactic factors has been studied in chambers divided into upper and lower compartments by a membrane through which cells can pass [l-4]. Cells in the upper chamber respond to chemotactic stimuli in the lower chamber by migrating through pores in the membrane and spreading on its under surface. The magnitude of This work was supported by the MRC of Great Britain. the response is quantified by a count of the number of cells/unit area of membrane. We found that mouse peritoneal macrophages References did not respond to chemotactic stimuli in 1. Thomas, DB, Nature 233 (1971) 317. 2. Lamont, J T, Perotto, J L, Weiser, M M & Is- the lower chamber unless serum was added selbacher, K J, Proc natl acad sci US 71 (1974) to the tissue culture medium in which the 3726. 3. Boyd, R L, Rolland, J M & Couchi, M N, Imm cells were suspended. We now show that a comm 3 (1974) 337. single serum protein is responsible for this 4. Schimnacher, V & Festenstein, H, J imeffect. Furthermore, the action of the promunogenetics 2 (1975) 337. 5. Robinson,PJ&Roitt,IM,Nature250(1974)517. tein is reflected not only in chemotactic 6. Robinson, P J, Penfold, P L & Roitt, I M, Clin exp responses, but in enhanced macrophage immuno123 (1976) 98. 7. Allen, A K, Neuberger, A & Sharon, N, Biochem j movement in the absence of a chemotactic 131(1973) 155. 8. Gybngybssy, M I C & Playfair, J H L, Cell im- stimulus and in marked spreading on Petri munol7 (1973) 118. dish surfaces. Exp Cell Res 102 (1976)