Cell Biology and Immunology of Leukocyte Function

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CHAPTER I

LYMPHOCYTE ACTIVATION

Outline

Chapter 1: THE LYMPHOCYTE MEMBRANE AND THE CYTOSKELETON

Chapter 2: TRIGGERING SIGNALS FOR T-CELL ACTIVATION

Chapter 3: STUDIES OF LYMPHOCYTE ACTIVATION

Chapter 4: FUNCTIONAL ORGANIZATION OF THE LYMPHOCYTE PLASMA MEMBRANE RELATING TO CELL ACTIVATION

Chapter 5: INVESTIGATIONS ON THE ROLE OF CA2+ AS A POTENTIAL SECOND MESSENGER FOR T AND B LYMPHOCYTE ACTIVATION AND ITS RELEVANCE IN DNA SYNTHESIS

Chapter 6: A MODEL FOR THE CONTROL OF POTASSIUM TRANSPORT IN PHA-STIMULATED HUMAN BLOOD LYMPHOCYTES

Chapter 7: EFFECT OF CON-A ON THE CATION AFFINITY OF THE Na+- K+ PUMP IN LYMPHOCYTES

Chapter 8: ACTIVATION OF LYMPHOCYTES WITHOUT MITOGENS BY INCREASING THE CALCIUM CONCENTRATION IN THE CELL CULTURE MEDIUM

Chapter 9: MEMBRANE MECHANISMS IN LYMPHOCYTE ACTIVATION WORKSHOP SUMMARY

Chapter 10: SYNTHESIS AND PROCESSING OF RNA IN STIMULATED FIBROBLASTS AND LYMPHOCYTES

Chapter 11: REGULATION OF PROTEIN SYNTHESIS DURING LYMPHOCYTE ACTIVATION BY PHYTOHAEMAGGLUTININ

Chapter 12: EVIDENCE FOR AND AGAINST UNUSUAL SPECIES OF DNA IN LYMPHOCYTE ACTIVATION

Chapter 13: STUDIES ON mRNA IN RESTING AND GROWING LYMPHOCYTES

Chapter 14: V- AND C-PARTS OF IMMUNOGLOBULIN n-CHAIN GENES ARE SEPARATE IN MYELOMA

Chapter 15: EVIDENCE FOR A NON-MESSENGER RNA WHICH IS RATE LIMITING FOR PROTEIN SYNTHESIS IN RESTING LYMPHOCYTES

Chapter 16: ADENYLATE CYCLASE IN NORMAL AND LEUKEMIC HUMAN LYMPHOCYTES

Chapter 17: ON THE COMMITMENT OF LYMPHOCYTES TO DNA REPLICATION: A POSSIBLE ROLE FOR TRANSGLUTAMINASE

Chapter 18: COOPERATION REQUIREMENTS OF MITOGEN STIMULATED LYMPHOCYTES IN AGAROSE-GEL CULTURES

Chapter 19: LECTIN-BINDING SURFACE PROTEINS OF HUMAN T-LYMPHOCYTES. A COMPARISON BETWEEN MITOGENIC AND NONMITOGENIC LECTINS

Chapter 20: PARALLEL INDUCTION OF T-CELL STIMULATION: POSITIVE CONTROL OF T-CELL RESPONSE BY MITOGEN TREATED MACROPHAGES

SECTION 1

PLASMA MEMBRANE STRUCTURE AND FUNCTION

Outline

Chapter 1: THE LYMPHOCYTE MEMBRANE AND THE CYTOSKELETON

Chapter 2: TRIGGERING SIGNALS FOR T-CELL ACTIVATION

Chapter 3: STUDIES OF LYMPHOCYTE ACTIVATION

Chapter 4: FUNCTIONAL ORGANIZATION OF THE LYMPHOCYTE PLASMA MEMBRANE RELATING TO CELL ACTIVATION

Chapter 5: INVESTIGATIONS ON THE ROLE OF CA2+ AS A POTENTIAL SECOND MESSENGER FOR T AND B LYMPHOCYTE ACTIVATION AND ITS RELEVANCE IN DNA SYNTHESIS

Chapter 6: A MODEL FOR THE CONTROL OF POTASSIUM TRANSPORT IN PHA-STIMULATED HUMAN BLOOD LYMPHOCYTES

Chapter 7: EFFECT OF CON-A ON THE CATION AFFINITY OF THE Na+- K+ PUMP IN LYMPHOCYTES

Chapter 8: ACTIVATION OF LYMPHOCYTES WITHOUT MITOGENS BY INCREASING THE CALCIUM CONCENTRATION IN THE CELL CULTURE MEDIUM

Chapter 9: MEMBRANE MECHANISMS IN LYMPHOCYTE ACTIVATION WORKSHOP SUMMARY

THE LYMPHOCYTE MEMBRANE AND THE CYTOSKELETON

Francis Loor,     Basel Institute for Immunology, Basel, Switzerland

Publisher Summary

This chapter focuses on the lymphocyte membrane and the cytoskeleton. Biochemistry and immunofluorescence studies have shown that the lymphocyte contains all the various structural and mechanochemical proteins known to constitute the cytoskeleton of most eukaryotic cells, that is, tubulin, actin, myosin, α-actinin, and so on. In the resting lymphocyte, thin section electron microscopy shows a narrow, dense network of thin microfilaments (MF), probably made of actin. Myosin is present in the cortical cytoplasm but does not seem to be organized as thick filaments. Microtubules (MT) are frequent. Most MT are connected with the centrioles. Some MT run parallel to the plasma membrane (PM) but remain separated from it by the MF network. Few MT are oriented toward the PM and terminate at it or close to it. Thus, there can be many more direct interactions between MF and PM than between MT and PM. Membrane component clustering by external, cross-linking ligands suggests membrane fluidity. Clustering is a prerequisite for capping to occur. Extensive crosslinking of membrane glycoproteins by some lectins inhibits capping.

INTRODUCTION

Structural and physiological aspects of the lymphocyte surface have recently been a subject of several reviews (1–4). Lately, a number of important observations have still appeared. Since the first description of membrane component spotting and capping by external ligands, and the original proposal of the fluid mosaic membrane model, our understanding of lymphocyte surface organization has significantly changed and the models need revision. In particular, they have to fit the important fact that cytoskeletal structures influence the organization of the lymphocyte surface, namely the expression of microvilli (MV) and the formation of the cap and of the uropode. The experimental evidence comes from morphological observations with the fluorescence and electron microscopes, from the use of drugs which disorganize the cytoskeleton, and, more recently, from the biochemical analysis of lymphocyte membrane components. The new concept of plasma membrane (PM) organization constitutes the central part of this paper. I shall discuss both the model, its bases and its implications in extenso elsewhere (Loor, manuscript in preparation).

A SUMMARY OF THE DATA

Biochemistry and immunofluorescence have shown that the lymphocyte contains all the various structural and mechanochemical proteins known to constitute the cytoskeleton of most eukaryotic cells, i.e. tubulin, actin, myosin, α-actinin, and so on. In the resting lymphocyte, thin section electron microscopy shows a narrow, dense network of thin microfilaments (MF), probably made of actin. Myosin is present in the cortical cytoplasm but does not seem to be organized as thick filaments. Microtubules (MT) are frequent. Most MT, perhaps all, are connected with the centrioles. Some MT run parallel to the PM, but remain separated from it by the MF network. Few MT (a maximum of about a hundred per lymphocyte) are oriented towards the PM and terminate at it or close to it (1–4). Thus, there can be many more direct interactions between MF and PM than between MT and PM.

Typical studies on the capping phenomenon have shown the following (1–4). Membrane component clustering by external, cross-linking ligands suggests membrane fluidity. Clustering is a prerequisite for capping to occur. Extensive cross-linking of membrane glycoproteins by some lectins inhibits capping. Capping requires active metabolism. Capping is inhibited by agents which interfere with MF function, e.g. cytochalasins. If anything, depolymerization of MT, e.g. by drugs such as colchicine and vinblastine, helps capping? in fact, it even relieves from the inhibition of capping caused by high doses of lectins. Capping occurs on the uropode. One finds an accumulation of actin, tubulin, myosin, MF and MT under the cap. Actin and myosin are clustered as sub-patches in the submembranous cortical cytoplasm, under the patches of whatever clustered PM component, (e.g. ref. 5). Thus, capping appears as a contractile phenomenon, mediated by MF, with MT playing essentially an antagonistic role, though some MT may also polarize MF activity (1–4).

Independently from those studies on capping, other characteristics of the cell surface were described (1–4). The lymphocyte surface is not always smooth but can be covered by multiple MV. In the case of the motile lymphocyte, the uropode is studded with MV, the rest of the cell body is smooth. MV expression is abolished by cytochalasins. Each MV, whether on the cell body or on the uropode, contains a bundle of MF. Uropode formation requires active metabolism and intact MF function (6). Migration of MV, and of their MF backbone, to the uropode is favoured or even induced by MT depolymerisation (7). Metabolism inhibition provokes an increase in MV expression on the cell body (1). Lymphocytes labelled with an external ligand may show MV, spots, PM invaginations or pinocytosis vesicles, depending on the ligand and on medium conditions (8). Polar accumulation of PM pinocytosis vesicles with membrane bound ligand requires active metabolism and intact MF (1,8). Thus, MV and PM pinocytosis vesicles are under a cytoskeletal control, some features of which are reminiscent of the capping phenomenon (1,8).

Two more observations now appear crucial in the context of the new model of cell surface organization. On B lymphocytes with a smooth surface, membrane immunoglobulins (mIg) are homogeneously distributed, whereas on villous B cells, mIg is at much higher density on MV than on the cell body (up to 6 times) (8–10). In leukocytes, PM components which are involved in nucleoside and amino acid transport, are not endocytosed during the process of phagocytosis, e.g. of PM glycoproteins clustered by lectins, but they are internalized when MT are disrupted by drugs (11,12). Thus, there is a definite, nonuniform distribution of some membrane components, which assume essential metabolic functions or recognition functions. These essential data and others to be discussed in detail elsewhere suggest the following model.

THE ORGANIZATION OF THE PLASMA MEMBRANE: A MOSAIC OF FLUID DOMAINS

The PM is not a homogenous fluid mosaic of proteins and lipids; some membrane components have a cytoskeleton controlled location. Thus, PM components fall into three distinct classes: I, II and III. The large majority of PM components (class I) are entirely free to diffuse in the plane of the membrane. Some PM components (class II) are anchored to MF, either directly or via intermediate pieces (e.g. α-actinin); they have practically no freedom, unless MF depolymerize or dissociate from the PM. Such components thus show a special affinity for MF and follow continuously their movements on the cell surface, e.g. cycling from one pole to another. Some other PM components (class III), less frequent, are anchored to the few MT which terminate at the PM, possibly via intermediate pieces such as the MT cross-bridges molecules. They show only the very restricted mobility typical of MT and a very limited freedom, unless MT depolymerize.

Class I components are ubiquitous on the PM, but class II and class III components can be essentially sequestered in different, restricted areas of the cell PM. This therefore delimits different domains on the PM: of type I, II or III. Domains of type I are a pure fluid mosaic; domains of type II can expand or narrow depending on the MF activity associated with MV, PM invaginations and pinocytosis vesicles formation; domains of type III are stable, being associated with MT; they always remain on the cell body, being neither expressed on MV nor included in pinocytosis vesicles.

An important remodelling of the cell surface occurs during an activation of MF activity (e.g. MV expression, formation of pinocytosis vesicles), which perhaps corresponds to cell activation. There is a concomitant phase separation of the membrane lipids into more fluid and more viscous areas (possibly, there is only increased segregation). Class III PM components (MT associated) constitute, because of their very restricted mobility, some kind of nucleating centers for the more viscous membrane lipids, while the more fluid lipids tend to associate with rapidly moving class II PM components (MF associated). Class I PM components function as a diluent which can be included in - or excluded from domains II and III depending on the particular circumstances.

With regard to the nature of the different PM components, class I components can be anything. Class II and class III PM components have to associate with MF or with MT and, as a minimum requirement, they should be integral proteins on the inner face of the PM. It would be even better if they were transmembrane glycoproteins, since the presence of a strongly hydrophilic domain (e.g. a large carbohydrate group) exposed on the outer face of the PM would constitute a perfect lock to keep such components in the PM. Since diffusion rates of membrane lipids are 10-100 times higher than diffusion rates of membrane proteins (1–4), membrane lipids, together with class II and class III membrane proteins, are the main cause of the separation of the PM into domains. Other membrane proteins will then segregate, by moving in or out of the different domains depending on their physicochemical characteristics.

A COROLLARY OF THE MODEL: THE CELL SEGREGATES METABOLISM AND RECOGNITION

A segregation of the PM components into different domains may appear unexpected to occur for a little round cell such as the lymphocyte. Hence, there is no doubt that in other cell types, such a segregation exists: it occurs in most cells which are organized as a tissue, such as epithelial cells, but also for some single cells, such as the sperm cell. Such cells show a high asymmetry and a high specialisation of the different membrane compartments which have to assume different specific functions.

What kind of segregation could occur for the lymphocyte PM? I suggest that the recognition functions of the cell membrane are associated with the labile domains of type II, while the essential metabolic functions of the cell PM are associated with the more stable domains, of type III. Further the lymphocyte could, either spontaneously or in response to various environmental stimuli, increase the segregation of domains II and III, by means of increased interactions of MF with type II PM components and of increased stabilisation of domains of type III by MT. There are some data to support such a concept. Thus, for lymphocytes, molecules having a recognition function such as mIg are at higher density on MV (type II domain) (8–10); mitogenic doses of lectins increase both MV formation by actin polymerisation into MF (1,8,13) and stabilization of type III domains by tubulin synthesis, MT polymerization and increased MT attachment to PM (14–17); encounter with particulate antigens, such as erythrocytes, increases MV formation (1); and so on. In other small rounded cells, the polymorphonuclear leukocytes, membrane sites essential for the cellular metabolism are not phagocytosed, unless the cells were treated with colchicine (11,12); encounter and phagocytosis of particules normally results in increased MT polymerisation (18) and this may be to better stabilize type III domains and avoid their internalization. Finally, one of the multiple reasons why lymphocyte stimulation by lectins is slowed down by colchicine (see ref. 19) could be that an efficient structural organization of an enzymatic chain or whatever metabolic unit in type III domains is lost, making the sequence of biochemical interactions less efficient (20).

MECHANISM OF CAPPING

All types of redistribution of sections of the PM toward one cell pole (MV, real two-dimensional spots, PM invaginations still open to the outside, and pinocytosis vesicles) are due to their dragging by MF, some MT playing, possibly, the role of a guide to polarize the general movement of MF.

When free PM components (class I) are cross-linked by external ligands into large clusters which include MF anchored sites (class II), such clusters will follow the movement of the MF bundles toward the cell pole. If MT associated sites (class III) are taken in the clusters too, they will directly interfere with the normal capping. They may be brought together with their associated MT into the cap or they may block capping, e.g. the blockage of capping by high doses of lectins (1–4) which induce a high increase in cytoplasmic MT by tubulin polymerisation (14–16). The result will depend on the particular balance of MT and MF in that cell and on MT orientation. Probably not all MT of the lymphocyte are oriented from one cell pole to the other, to polarize MF movements, and some cytoplasmic MT must continuously antagonize such MF movements. The natural tendency of the MF bundles to contract into the cell uropode is revealed by the fact that such spontaneous redistribution is readily obtained simply by treating the cells with drugs which depolymerize MT (7). This is, however, a truly spontaneous process observed for lymphocytes which form a uropode and start moving (21,22). Depolymerisation of antagonistic MT is probably regulated by the cell in a precise way. This may be achieved by specific proteins regulating local Ca²+ levels or by cyclic nucleotides, unless the higher activation of MF or constitution of bundles of MF were sufficient to cross a MT barrier that would have thixotropic properties (1).

MICROVILLI-MEDIATED RECOGNITION

There is a substantial evidence that intercellular recognition in a variety of cell types is mediated by virtue of MV. Lymphocytes too use MV to establish contact with other lymphocytes or with other types of cells (1). This is based both on morphological observations with the optical and electron microscopes, and on the effects of drugs which interfere with the cytoskeleton on such recognition. Lymphocyte MV, either distributed on the whole cell or concentrated on the uropode, are involved in the following types of intercellular interactions: the spontaneous and lectin-induced aggregation of lymphocytes; the constitution of the nonimmune specific T cell rosettes; the migration of lymphocytes from the blood to the lymphoid tissue, especially the recognition of the high endothelial cells of the post capillary venules; various systems of killer-target cells interactions, especially the recognition phase; the antigen-specific macrophage-lymphocytes interactions. A detailed analysis of the involvement of the cytoskeleton in such interactions will appear elsewhere. Finally, a possible mechanism for shedding consists in release by the cell of small bits of PM by pinching off MV tips, and there is good evidence to support such a suggestion (1). According to our model, only class II domains would be shed, while important PM metabolic units would not.

CONCLUSIONS

The essential feature of this model of cell surface organization is the existence on the plasma membrane of domains specialized in functions of recognition and other domains concerned with cell metabolism. Though there must be some interdomain interactions, these two types of domains appear as essentially autonomous.

REFERENCES

1. Loor, F. Prog. Allergy. 1977; 23:1.

2. de Petris, S.Nicolson, G.L., Poste, G., eds. Cell Surface Reviews; 3. North-Holland-Elsevier, Amsterdam, 1977:643.

3. Nicolson, G.L. Biochem. Biophys. Acta. 1976; 457:57.

4. Schreiner, G.F., Unanue, E.R. Advan. Immunol. 1976; 24:37.

5. Bourguignon, L.Y.W., Singer, S.J. Proc. Nat. Acad. Sci. USA. 1977; 74:5031.

6. de Petris, S. J. Cell Biol. 1978; [in press].

7. Albertini, D.F., Berlin, R.D., Oliver, J.M. J. Cell Sci. 1977; 26:57.

8. Loor, F., Hägg, L.-B. Eur. J. Immunol. 1975; 5:854.

9. Linthicum, D.S., Sell, S. J. Ultrastructure Res. 1975; 51:55.

10. de Petris, S. Nature. 1978; 272:66.

11. Ukena, T.E., Berlin, R.D. J. exp. Med. 1972; 136:1.

12. Oliver, J.M., Ukena, T.E., Berlin, R.D. Proc. Nat. Acad. Sci. USA. 1974; 71:394.

13. Criswell, B.S., Rich, R.R., Dardano, J., Kimzey, S.L. Cell. Immunol. 1975; 19:336.

14. Edelman, G.M., Wang, J.L., and Yahara, I., Cold Spring Harbor Conference on Cell Proliferation, Vol. 3, p. 305 (1976).

15. Hoffstein, S., Soberman, R., Goldstein, I., Weissman, G. J. Cell Biol. 1976; 68:781.

16. Oliver, J.M., Albertini, D.F., Berlin, R.D. J. Cell Biol. 1976; 71:921.

17. Sherline, P., Mundy, G.R. J. Cell Biol. 1977; 74:371.

18. Burchill, B.R., Oliver, J.M., Pearson, C.B., Leinbach, E.D., Berlin, R.D. J. Cell Biol. 1978; 76:439.

19. Wang, J.L., Gunther, G.R., Edelman, G.M. J. Cell Biol. 1975; 66:128.

20. Greene, W.C., Parker, C.M., Parker, C.W. J. Immunol. 1976; 117:1015.

21. Schreiner, G.F., Braun, J., Unanue, E.R. J. exp. Med. 1976; 144:1683.

22. Schreiner, G.F., Fujiwara, K., Pollard, T.D., Unanue, E.R. J. exp. Med. 1977; 145:1393.

DISCUSSION

Unidentified speaker

Would you like to speculate that capping is mediated by myosin?

Loor, Basel

It is evident that this whole model of capping is thought to be a contractile event and it would be easy if it were similar to the sliding filament model of muscle contraction. However, myosin does not seem to be organized as thick filaments in lymphocytes. It may exist as single molecules or as submicroscopical structures. I could not tell where the myosin, which is present in the lymphocyte, should be placed in the model.

Ashman, Los Angeles

Would you comment on evidence relating microfilaments to capping and cytochalasin B effects. Cytochalasin B alone may not inhibit capping.

Loor

Cytochalasin B in my experience is the worst of all cytochalasins to be used to inhibit capping. Cytochalasin A is the best inhibitor of the process. To obtain a good inhibition of capping by cytochalasins one needs to preincubate the lymphocytes for some time (10-15 min) at 37°C with the cytochalasins, probably to allow transport of the drug in the cells. Only then can one obtain a good inhibition. In that respect, I should recall the observation of a synergy between colchicine and cytochalasin to block capping. The usual interpretation is that both microtubules and microfilaments are involved in the dragging of aggregated material to the cap. A possible role for microtubules could be to constitute a guide to polarize a microfilament contractile activity. However, another interpretation is that there is better permeation of cytochalasin resulting from purely membranous effects of colchicine. Unfortunately the data of Unanue and of de Petris cannot exclude such a possibility, as these authors did not make use of the lumicolchicine control in this particular series of experiments (which has similar membrane effects as colchicine, but no effect on microtubules).

TRIGGERING SIGNALS FOR T-CELL ACTIVATION

A. Novogrodsky, A.L. Rubin and K.H. Stenzel,     Rogosin Kidney Center, Departments of Biochemistry and Medicine, Cornell University Medical College, New York, New York

Publisher Summary

This chapter describes triggering signals for T-cell activation. The lymphocyte membrane serves as a transmitter of external signals that induce cell proliferation and differentiation. The signals that interact with the cell membrane and trigger these events may be either immunologically specific and may induce a monoclonal proliferation or may be immunologically nonspecific and may induce a polyclonal proliferation. Most T-cell mitogens, agents that induce polyclonal proliferation of T-cells, interact with cell surface polysaccharide moieties. These mitogens include certain lectins and the oxidizing agents sodium metaperiodate and galactose oxidase. The initial step in the triggering event is mitogen binding to, or alteration of, a cell-surface saccharide structure. The nature of this interaction is central to the understanding of the molecular mechanisms responsible for lymphocyte activation. The chapter also describes the structural aspects of the mitogenic site. Although polyclonal agents appear to trigger cells to divide by affecting similar metabolic pathways, different agents selectively affect responses induced by different mitogens.

The lymphocyte membrane serves as a transmitter of external signals that induce cell proliferation and differentiation. The signals that interact with the cell membrane and trigger these events may be either immunologically specific, and induce a monoclonal proliferation, or may be immunologically non-specific, and induce a polyclonal proliferation. Most T-cell mitogens, agents that induce polyclonal proliferation of T-cells, interact with cell surface polysaccharide moieties. These mitogens include certain lectins and the oxidizing agents sodium metaperiodate (IO−4) and galactose oxidase (GO). The initial step in the triggering event is mitogen binding to, or alteration of, a cell surface saccharide structure. The nature of this interaction is central to the understanding of the molecular mechanisms responsible for lymphocyte activation.

STRUCTURAL ASPECTS OF MITOGENIC SITES

An oligosaccharide, common to many glycoproteins, can serve as the target for most if not all, T-cell mitogens. Figure 1 depicts a glycopeptide isolated from porcine thyroglobulin (1) and indicates how this structure can provide the target for a number of T-cell mitogens. For reasons that are not readily apparent, wheat germ agglutinin (WGA) was originally reported to be non-mitogenic (2). Recent studies have, however, clearly demonstrated its mitogenic properties (3). Hepatic binding protein (HBP), a protein that specifically binds asialoglycoproteins (4), has recently been found to be mitogenic for desialyated T-cells (5). To our knowledge this is the first mammalian lectin that has been found to have mitogenic properties.

FIGURE 1 Accommodation of T-cell mitogens on a single oligosaccharide moiety.

The fact that these mitogens can be accommodated in a single megalo-site does not necessarily indicate that there is a unique mitogenic site on the cell surface. Different glycoproteins share similar oligosaccharide structures, and the possibility that different mitogens induce blastogenesis by interacting with different glycoproteins should also be considered. Co-capping studies, however, indicate that several T-cell mitogens with different saccharide specificities interact with membrane sites that are physically linked (6).

The unique role of saccharide groups as targets for mitogens was substantiated further by studies involving the conjugation of a ligand to the cell surface, followed by assessing the mitogenic activity of the anti-ligand. Biotin does not serve as a mitogenic site for avidin when conjugated to cell surface protein moieties but only when it is conjugated to saccharide groups (7). Thus, binding of a lectin to a saccharide group per se is not a requirement for mitogenicity, since non-saccharide moieties can serve as mitogenic sites provided they are conjugated to a cell surface saccharide. It would appear that the saccharide moiety functions to provide a binding site for lectins at a critical location on the cell surface.

VALENCY OF MITOGENS

Does simple binding to critical surface sites suffice to induce blastogenesis, or is the triggering signal associated with cross-linking of surface sites? Cross-linking can only be directly imposed on cells by agents that have more than one binding site, that is, multivalent agents. Table I summarizes some data on the effect of the valency of mitogens on their blastogenic activity. In general, most studies have found polyvalency to be a prerequisite for mitogenic activity. Divalent fragments of anti-lymphocyte or anti-immunoglobulin antibody, for instance, are mitogenic, whereas the univalent fragments are not (8,9). Ravid et al. (10) have clearly shown that the divalent anti-DNP fragment is mitogenic for DNP conjugated cells, whereas the monovalent fragment is inactive. In some cases lectins fail to stimulate cells, even though they are divalent, and mitogenicity is seen only with polymeric preparations (11,12,13).

TABLE 1

Relationship of Mitogen Valency to Mitogenic Activity

Nevertheless, several reports have appeared indicating that blastogenesis is induced by monovalent antibody (14,15) and lectin (16). Several possibilities could explain these findings. The mitogen could interact with accessory cells via non-saccharide binding sites thus rendering these cells supermitogens with multi-valency. Alternatively, the mitogen could interact with additional, non-saccharide sites on the same cell to form a cross-linked structure. A hydrophobic binding site for Con A has in fact been shown (17).

COVALENT CROSS-LINKING AND MITOGENESIS

The possibility that covalent cross-linking of membrane sites might be related to mitogenesis should also be considered. We recently found that both Con A and PHA increased lymphocyte transglutaminase (TGase) activity (18). This enzyme is known to induce cross-linking of protein molecules via γ-glutamyl-ε-lysine bridges (19,20), TGase activity is present in human peripheral lymphocytes and is enhanced up to 15-fold within 10-30 min after treatment of the cells with Con A or PHA. The enzyme is not detected when intact cells are assayed; it is detected only in cell lysates. Con A enhances TGase activity only when it is incubated with intact cells; Con A treatment of cell lysates has no effect, αMM specifically inhibits the enhancement of TGase by Con A. Thus, the enhanced TGase activity in cells treated with Con A results from the specific interaction of the lectin with its saccharide binding site on the cell surface, rather than by direct interaction with the enzyme itself. The increased activity of TGase in cells treated with Con A, as compared to unstimulated cells, is maintained under assay conditions in which saturating levels of Ca²+ are present. TGase may therefore be involved in early cellular events leading to lymphocyte blastogenesis. This possible relationship is discussed in another paper in this volume (21). Taken together, the findings discussed above have led us to postulate a scheme that relates non-covalent to covalent cross-linking induced by lectins (Figure 2). The sites that may be covalently cross-linked are not necessarily the same as the lectin binding sites, even though they appear so in this diagramatic presentation.

FIGURE 2 A schematic relationship between non-covalent and covalent cross-linking of membrane sites. The lectin binding sites and covalently cross-linked sites may not neccessarily be the same (see text).

CELL-CELL INTERACTIONS IN TRIGGERING T-CELL ACTIVATION

Cellular interactions, in addition to cross-linking of membrane sites, appear to be important in the activation process. Blastogenesis induced by the mitogenic oxidizing agents provides a useful system for the study of cell-cell interactions in lymphocyte activation. The oxidizing mitogens induce blastogenesis by the generation of aldehyde moieties on cell surface glycoproteins (22,23,24). Aldehyde modified T-cells undergo blastogenesis when cultured with unmodified macrophages, and untreated T-cells undergo blastogenesis when cultured with aldehyde modified macrophages (25,26,27). It has also been shown that unmodified lymphocytes undergo blastogenesis when mixed with aldehyde modified lymphocytes that had been irradiated or treated with mitomycin (28,29). Macrophages are also required for blastogenesis in this latter system. Evaluation of the cellular interactions involved in blastogenesis induced by oxidizing agents is possible since excess oxidizing reagent can easily be removed after brief incubation, and the cells can be cultured in a mitogen free environment. Although the mechanism of aldehyde induced blastogenesis is not known, it has been postulated that cell surface aldehydes might react with amino groups to form Schiff bases (24). These interactions could lead to the formation of a cross-linked structure. Borohydride reduction of aldehyde modified cells has been found to inhibit blastogenesis. This inhibition could result from reduction of the aldehyde moiety with consequent interference with a cross-linked structure. Alternatively, inhibition could result from reduction of a Schiff base to a covalently cross-linked structure, a condition that could interfere with the progression of blastogenesis (Figure 3). A cross-linked structure mediated through Schiff base formation could occur either on the aldehyde modified cell itself, or between adjacent cells. The latter condition would provide a molecular basis for cell-cell interaction. However, another possibility should be considered. Cells treated with mitogenic oxidizing agents form dense aggregates by a time and temperature dependent process that also depends on active metabolism. This finding raises the possibility that cell-cell interactions induced in this system are not a direct result of aldehyde modification or Schiff base formation, but rather result from a secondary process stimulated by surface oxidation. This secondary process could be the generation of a cell-surface site that interacts with receptors on adjacent cells and thereby promotes blastogenesis. Thus, cross-linking of sites on individual cells could provide the first signal for lymphocyte activation, and this could lead to the generation of new cell surface structures. These new structures would then constitute the second signal and be dependent on cell-cell interactions (Figure 4). Kinetically, the first signal corresponds to a mitogen dependent phase of blastogenesis and the second signal to a phase that is mitogen independent. Similar mechanisms could also be postulated for lymphocyte activation induced by lectins. Analysis of lymphocyte activation involving lectin stimulation is, however, hampered by both the difficulty in removing lectin and by the propensity of the lectin itself to aggregate cells.

FIGURE 3 Potential chemical effects of the mitogenic oxidizing agents on cell surface groups and their reduction by borohydride.

FIGURE 4 Dependency of blastogenesis on cell-cell interactions: A proposed two signal model for lymphocyte activation (see text).

SELECTIVE EFFECTS ON BLASTOGENESIS INDUCED BY DIFFERENT MITOGENS

It should be noted that, although polyclonal agents appear to trigger cells to divide by affecting similar metabolic pathways, different agents selectively affect responses induced by different mitogens. We recently found, for instance, that colchicine has differential effects on blastogenesis induced by different stimuli (30). Proliferation of human lymphocytes induced by I0−4 is potentiated by a 30 min exposure to colchicine (10−6M), followed by washing to remove excess reagent, whereas the response to Con A under similar conditions is inhibited. If colchicine is allowed to remain in the culture media, responses to both agents are severely depressed. Treatment with colchicine before or after IO−4 modification has similar enhancing effects. Lumicolchicine (a photo-inactivated derivative of colchicine that does not disrupt microtubules) does not alter proliferative responses. In addition to the proliferation of IO−4-oxidized cells, irradiated IO−4-modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO−4-modified cells are treated with colchicine. Proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of a brief treatment of cells with colchicine on lymphocyte responses induced by cell-cell