Intercellular Interactions in Eukaryotic Homeostasis

REVIEWS

Intercellular Interactions in Eukaryotic Homeostasis M. K. Agarwal I N S E R M U-36, 17 r u e d u Fer-A-Moulin, 75005 Paris, France

Introductory remarks

As opposed to the prokaryotic cell which must adapt its intracellular processes to face the challenge posed by changing environments, Eukaryotic cell needs to contribute its share to maintain constancy of It. milieu interieur. Indeed, the very concept of homeostasis calls for coordination within and between the various tissues and organs towards realization and expression of pre-determined reaction patterns. Perhaps the most characteristic feature of a neoplastic cell is acquisition of an autonomy over the morphofunctional limits imposed by the microenvironment in a cellular community. This reasoning may easily be used to explain the autonomous sort of transformation exhibited by normal cells in culture. On the other hand, development is believed to require cell-cell flow of ‘morphogens’ at varying distances from the morphogen source,*3 thereby commiting the cellular communities to restraints dictated by predetermined differentiation patterns. 111 the integrated organism, various levels of communication may be distinguished that ensure this functional co-operation between the component parts. At the subcellular level, the nucleo-cytoplasmic exchange of information is obviously well established.17 Between adjacent cells, direct cell-cell interactions may be mediated either by modification of cellular membranes, or through intercellular junctions. Continuity of conversation between cells located at some distance is assured by chemical messengers in the form of hormones, vasoactive or chemotactic substances. Here, the extracellular compartment plays the necessary role to co-ordinate contact between the control-initiating and sensitive cells, as well as the organs that respond to the bioactive substance via retroactive adjustments in various metabolite pools. Apart from these levels of control, a Difierentiurion 2, 1974

phenomenon such as cellular co-operation requires successive participation of various distinct cell types to obtain completion of a single biological event, viz: immunogenesis. In its simplest form, the question may be asked whether such co-operation is called for in other types of biological processes, and in various stages of differentiation; and whether these systems would share some analogy. This is the focal point of the present review, although selected other systems have also been included in the perspective. After a brief survey of instances where this type of intercellular co-operation has been documented or indicated, efforts are made to trace a possible common origin that might explain a diversity of response patterns in various stages of differentiation. This review is not intended to be an exhaustive account of the literature on the various forms of cellular interactions mentioned above. Rather, relevant features from each system have been taken to arrive at a possible level of regulation which has hitherto remained elusive. The references are not meant to establish priorities but to lead the reader to an appropriate source reference. For these reasons, mention is often made to existing review articles rather than to descriptions of particular details.

Short range interactions Morphofunctional studies

Contact interaction may be studied by adhesion,6* exchange through intracellular clefts80 or passage from cytoplasm to cytoplasm via special membrane contacts.33,53,54 The cytoplasmic bridges provide the most complete means of cell interaction since many macromolecules and organelles may traverse such contacts.31 In the nervous system, short range secretions and 371

M. K. Agarwal membrane contacts are required for normal function. The low resistance junctions may be visualized under the electron microscope as a variety of morphological specializations, usually in characteristic combinations and locations in the particular type of embryonic or adult tissue. They have been classified into five major categories. In the chemical synapse, cell interaction is mediated by a special transmitter substance that diffuses across the cxtracellular fluid filling the synaptic cleft. These are highly specialized forms of junctions requiring a sequential and complex series of steps for the completion of cellular activity, and are adapted primarily to the conditions of adult life.48 Simple apposition between membranes of adjacent cells may act as a possible mechanism of cellular interaction especially during heavy activity, e.g. in nervous tissue where K + concentrations change markedly. Simple desmosomes and intermediate junctions (e.g. intercalated discs) maintain the mechanical integrity, at least in the heart.64 Septate desmosomes have been observed in non-vertebrate embryos. They are also conspicuously present in Dvosophilu salivary gland epithelium but their exact role is still uncertain.89 A different kind of low resistance junction resembles cytoplasmic bridges in that they permit exchange of selected cellular constituents, but differ from bridges by being present between quite dissimilar cells. Tn excitable cells, they are referred to as electrical synapses and represent the sites where potential changes spread passively and directly from one cell to another, as cvident from microelectrode penetration in the giant motor synapse of the crayfish. There is evidence that tight junctions may represent the site of ion transfer.33.69 Electric measurements and tracer analysis have revealed the presence of a system of passageways, connecting adjoining cells in most tissues, through which molecules of molecular weights as much as 104 have been recorded as passing under certain conditions. This communication is widespread in normal cells and in embryonic tissues and seems to be uncoupled in certain malignant epithelial tumours which fail to establish connections both between themselves as well as with normal cells.s3~s9 Abundance of such low resistance junctions in embryonic tissues indicates a possible integrative function but experimental proof is still wanting. In addition, they also develop between normal and cancerous cells in culture.33.69 In an entirely independent series of studies, electron micrographs of the human stomach have 312

revealed unusual, intimate relationship between endocrine cells and other epithelial cells; this was lacking in other gastric epithelial cells.72 This was suggestive o f some kind of close communication between selected cellular types. Biochemical studies

Mutant hamster cells, deficient in inosinic pyrophosphorylase (BHK-IPP-) or thymidine kinase (BHK-TK-), are able to incorporate hypoxanthine or thymidine, respectively, into nucleic acid only when they arc cultured in presence of the wild type BHK cells.68 This appears to be due to the transfer of nucleotides from the wild type to the mutant cell type, as is also evident from studies with human fibroblasts from normal or Lesch-Nyhan (IPP-) patients. Furthermore, BHK-IPP or BHK-TK- cells fail to grow separately in a medium containing both hypoxanthine or thymidine; however, in mixed, confluent cultures, both mutants grow at the rate of the wild type, presumably due to nucleotide exchange via long cytoplasmic extensions. This property of interaction is under genetic control. Thus, mutants (L-IPP- and L-TK ) derived from mouse cell line L 9 2 9 are interaction negative (int-) and maintain mutant phenotypes even in mixed cultures. However, hybridization of BHK-intwith L-int- cells by fusion with inactivated Sendai virus confers the interaction property on to L929 mutant cells. Interaction is not limited to cells in culture. Heterozygous mothers of Lesch-Nyhan children are clinically normal although they possess two genotypically distinct cellular populations.57 This sort of reasoning may explain the observation that mixing of only 10% of wild type D. discoideum cells permits development of fruiting bodies possessing spores of both parental types.28 Cells fused in vitro Much discussion has occurred on the specificity of cell aggregation palterns,22,27,3Y,70,*4,91contact inhibition of cells in c~lturel,24,34,44,61,62 and growth release by serum, AMP in such systems.11366 Factors responsible for these phenomena have been partially characterized.61,62,66,67 These facts would suggest that this sort of symbiotic homeostasis requires intercellular dialoguc initiated upon establishment of contact. Rather than developing these aspects, it is more pertinent for the purpose of this review to bricfly examine the model of cellular hybridization.

Differentiation 2, 1974

Cell-cell communications

The influence of intercellular interactions may be demonstrated by fusion experiments in vitru.13,25,30,37,86 Most cell types from a wide range of animal species, and showing very diverse forms of specialization, can be made to fuse in vitro in the presence of the inactive cell envelope of the Sendai virus.14J784o In certain combinations, chromosomes are eliminated in early cellular divisions, and this is common on prolonged cultivation in vitro. Usually, the karyotype consists of varying proportions of chromosomes from both parents. In such a system, it was demonstrated that the ability o f a hybrid to produce tumours was restricted to cells from which certain chromosomes had been eliminated. Moreover, a diploid fibroblast was able to suppress the malignant phenotype when a complete chromosome set of both parents had been retained. These differences were not explicable in immunological terms. Rather, the nonmalignant partner contributes some chromosomelinked factor(s) capable of checking the expression of malignancy of the parent tumour cell. In some cases, the malignant phenotype could be suppressed by fusion of two malignant partners. These results obviously demonstrate the importance of cellular interaction, whose loss in vivo would lead to selection, expression and proliferation of a neoplastic cell. More detailed and exhaustive accounts of cell fusion in vitru are available. 1 3 25,29,30 I

Long range interactions

The nervous system displays an extraordinary series of everchanging cell relationships. This may imply the existence of cell interactions capable of determining the eventual morphofunctional organisation in the nervous system.ls,92 In invertebrates, neurones and their target cells seem to be endowed with a mechanism(s) for recognizing their correct partners. Failing to obtain the normal axon, a target cell will accept innervation by a non-specific partner. However, when the normal partner becomes available the non-specific axon is inactivated and discarded.45 Studies on neural crest development in vertebrate embryos indicate that the pluripotent cell population migrates extensively and localizes precisely, possibly under the influence of morphogenetic stimuli, such as the nerve growth factor which may act through contact alterations between crest cells.87 Further evidence on intercellular communications comes from work on synaptic transmission in the leech. It has been shown that regeneration Differentiation 2, 1974

introduces compensatory changes in parts of the nervous system not directly affected by the surgical lesion.12 A genetic analysis of such relationships has recently become possible and several systems may be cited. The first example concerns the inbred RCS rats homozygous for the autosomal recessive gene(rd) for retinal degradation. Both the outer photoreceptor and the inner pigment epithelial cell arise from adjacent sectors of primitive neuroepithelium in the embryonic optic vesicle. In adulthood, Vitamin A derivatives shunt between these two cellular compartments when the eye is alternatively dark-adapted or exposed to strong light. In normal retinal development, the protein rich neuromembranes, synthesized and shed from the outer layer of photoreceptor cells, seem to be ingested by the inner layer of pigment epithelium.41 The rd/rd mutant epithelium does not engulf the membranous discs shed by the rod segment, although the phenotypic expression of the mutant involves degeneration of the photoreceptor cells.76 Electron microscopic autoradiography has further revealed that the mutant retina exhibits extra lamellar whorls, between the two cell layers, which are enriched in protein (rhodopsin ?) synthesized in the epithelial cell cytoplasm. In the second example, the stagger mutation (sg) in the mouse is expressed as a disorder in the interactions between the granule cell and the Purkinje cell neurone of the cerebellum. Normally the synapse between a granule cell axon and a Purkinje dendrite are the most readily visualized and abundant of the synapses in the cerebellar cortex. Loss of this particular intercellular contact in sg/sg mice (where all other synapses are normally present) results in degeneration and decimation of granule cell population leading eventually to cerebellar atrophy. All the evidence points to inability of the Purkinje cell to develop the dendrite spines that in normal animals become innervated by granule cell axons. From this, it follows that the Purkinje cell may noramlly influence cellular kinetics in the external granular layer; diminution of this influence would result in the observed early reduction in cerebellar size, and to increased number of pycnotic nuclei in the external granular layer. Furthermore, normal survival of maturing granular cells might be dependent on the establishment of specific contacts with Purkinje cells. Thus, it is felt that granule cell-Purkinje cell interaction is central both to the stagger problem and to a normal genesis of the cerebellar cortex.76 373

M. K. Agarwal The third system is comprised of the reeler (rl) mutation in mice which exhibit widespread derangement of cell relationships in cerebellar and cerebral cortices, leading to disturbed laminar organization and cell orientation patterns. Thus, in normal differentiation young neurones move outward from the site of origin by passing along radially oriented ‘guide’ fibres. In rl/rl mutants, cortical neurones are generated in normal locations at normal times, but fail to attain the usual laminar distribution. Furthermore, the early and the late generated cortical layers are more or less reversed in position, possibly due to abnormal distribution of early axon contacts. Such defects of intercellular contact are also present in reeler cerebellar cortex.76 At a chemical level, synthesis and inactivation of neurotransmitters (catecholamines) proceeds via mutual interactions between (soma and endings) and within the various cells involved in the same pathway.36 The selective destruction of adrenergic nerve endings in the peripheral nervous system by 6-hydroxydopamine, moreover, leads to a long lasting reduction in the activities of tyrosine hydroxylase and dopa decarboxylase in brain areas rich in catecholamine-containing nerve endings.85 These and various other examples36 suggest a chemotactic guidance system responsible for co-ordinating the morphofunctional homeostasis in the nervous system. A more characteristic account of such factors has recently been provided by study of embryonic brain cells in culture.34~61-63 A cell-free, homologous supernatant enhanced the aggregation of embryonic cerebral cells in a dose-dependent manner. This effect was not dependent upon the embryonic age of the aggregating cells, and the supernatant did not have such effects on cells from other areas of brain, or from non-nervous tissue. T h e phagocytic cells

The reticuloendothelial system (RES) is comprised of a number of distinct cellular types distributed in various tissues, and performing a variety of similar and related functions. The vascular endothelium, the Kupffer’s stellate cells lining the hepatic sinusoids, the lung and spleen macrophage, the brain glia cells, and the stem cells in the bone marrow, represent the fixed elements of the RES. Representative examples of the wandering or scavenger cells of the RES may be found in histiocytes, lymphocytes, macro374

phages, monocytes, polymorphonuclear leukocytes and neutrophiles. It is important to keep in mind that morphofunctional transformation within these cellular types is not infrequent. Besides, intercompartmental exchange between free and fixed cellular types occurs with considerable frequency. Thus, the RES (of mesenchymal or mesodermal origin) forms the prototype of a rich repertory where overall co-ordination and surveillance may be processed with much elaboration and ease (for a recent review see ref. 81). The most characteristic property of a number of RE cells resides in their capability to ingest or phagocytose substances in the colloidal range. Most often, this is followed by intracellular digestion, the degradation products being handled in various ways : simple elimination, immunogenesis (through participation of other types of RE cells), and possibly metabolic homeostasis (by epithelial cells). While analyzing these relationships, it should be recalled that in the course of evolution phagocytosis preceded other functions both in ontogeny and phylogeny. Thus, a primitive phagocytic function may be detected in the Molluscs but immunity was not visible before the Teleosts. Also, in mammalian differentiation, phagocytic property develops earlier than immunological competance. Therefore, it becomes important to appreciate whether an anomaly in one such integrated function is derived from a fault in the initial processing, or final expression, of events in an embryologically similar or different cell type, that may be localized either adjacent to or away from the primary target. Many examples of intercellular communications among RE cells may be cited. Cytological evidence has indicated bridge formation and the passage of bacteria and RNA between rabbit peritoneal histiocytes in diffusion chambers.10 Lung macrophages formad bridges both between themselves and with peritoneal histiocytes. Cell interaction has also been recorded between mouse leukaemia I ymphocytes and fibroblasts .43 When 3H-thymidine-labelledmouse leukaemic reticulum cells and L-strain cells were cultivated together for up to 13 days, hybrid cells (0.1 %) could be detected by autoradiography. The leukaemic cells not involved in hybrid formation were progressively lost by degradation and phagocytosis. Metabolic and regulatory functions of RE cells are variously known. Monocytes have been reported to control granulocyte proliferation and maturation.20 Lymphocytes may play a direct role in the carcinogenic process since they possess Diyerentiation 2, 1974

Cell-cell communications

a hydrocarbon hydroxylase necessary to both detoxify and activate polycyclic hydrocarbon (such as benzopyrene) to its potentially carcinogenic active form;** very numerous instances have been recorded of the important role of endothelial cells in physiological homeostasis, blood vessel permeability, response of blood vessels to physiological and pathological stimuli, thrombosis, arterosclerosis, and vasculitis.47 Furthermore, RES functional alterations are envisionned as regulating the growth process in somatic, regenerating and neoplastic liver, possibly by alteration in uptake and disposition of natural ‘Governor’ signals in the serum.79 This has been supported by recent work from our laboratory. Liver regeneration was accompanied by progressive activation of the R E function and hypertrophy of spleen - an organ rich in R E elements. With various combinations of cortisone, and a RE-active substance like endotoxin, it was shown that a temporal change i n the function of the R E cells did not parallel the behaviour of parenchyma after hepatic resection.3~4 Besides, a delayed burst of DNA formation was obtained in regenerating livers whose R E activity had been modified by administration of low doses of cortisone or endotoxin one hour after surgery. Under similar conditions, other RE-active agents like bentonite or celite also retarded the DNA synthetic response whereas latex particles, although engulfed by the Kupffer cells, did not alter either the R E activity or DNA synthesis. More important, partial hepatectomy did not, in mice exhibiting refractoriness to the noxious effect of endotoxin, initiate DNA synthesis, as it did in the control animals.5.7 These results could be most easily explained as demonstrating a possible regulatory influence of R E cells in the overall survey and co-ordination of cellular response (discussed more fully below). Hormone-specific gene modulation

The lyrnpholytic action of the glucocorticoid hormones (both in vivo and in isolated cells in vitro) may be contrasted with anti-anabolic hypertrophy effected by these steroids in various types of epithelial cells (liver or lung parenchyma). This point is often forgotten while analysing the nature of enzyme induction by glucocorticoids in intact organs. Although the normal hepatic lobule is comprised of only 30-40% littoral (RE) cells, both quantitative and qualitative adjustments in the available endothelial cell volume Differentiation 2, 1974

may be observed in certain pathophysiological syndromes. In our earlier studies it was shown that materials such as bacterial endotoxin, thorotrast and zymosan (which, because of their size, nature and properties, possess special affinity for the RES, and which, on microscopic or radioautographic examination, are present only in the RE cells of the liver) were nevertheless capable of influencing functions (e.g. enzyme induction by glucocorticoids) believed to be localized in the parenchymal cells.2.8 The influence of modification of R E function on the overall regenerative capacity of the liver was discussed above. Moreover, cortisone influenced DNA synthesis in normal, resting liver only if such processes (and RE function) had first been activated by an R E agent such as endotoxin or carbon.536 To account for these observations, it was proposed that R E cells may be involved in the overall co-ordination and expression of differentiated metabolic functions in liver parenchyma (and possibly elsewhere). The various messenger molecules produced by the RE cells and their possible influence on other sites, including enzyme induction, have already been discussed.2.8 This is supported by the fact that hitherto it has not been possible to maintain a functionally normal hepatic parenchymal cell in vitro. Where tested, such primary epithelial cells in culture do not respond to glucocorticoids by an elevation in enzyme or glycogen levels. The only (possible) exception to this comes from the induction by dexamethasone of tyrosine transaminase (TT) in hepatoma cell cultures.*3 This is highly unsatisfactory both because a natural steroid, cortisol, exhibits only limited effectiveness in this regard and because no attention has been paid to the isomorphic form46 of the TT protein induced under these conditions. The mechanism of this induction has been recently challenged.50 The processes regulating TT activity in vivo,moreover, are highly varied and do not seem subject to the type of co-ordinated regulation in various stages of cellular differentiation as is evident with other enzymes (e.g. tryptophan pyrrolase, phosphoenol pyruvate carboxykinase) that are specifically induced either by the hormone or the substrate.2 Thus, the hepatoma cell model cannot be considered a valid system to study the mechanism of hormone action as unfurled in vivo. It would be of more than passing interest to observe the behaviour of mixed endothelial-parenchymal cell cultures or of hybrids from these two cellular types. Efforts may also be made to observe the reaction pattern of parenchymal cells cultured 375

M. I(. Agarwal with RE cell extracts. Allophenic mosaicss8 offer another possibility to study such interactions.

For more exhaustive and recent reviews on B-T interaction see references.16,1*,42,49,71,75

lmmunogenesis

Organogenesis

Cellular interactions are known to occur at various points in the immune response, and these have been reviewed on many occasions. An overall analysis of these interactions, however, is always a difficult task due primarily to the rapid advances as well as the development of specialized techniques and heterogenous systems utilized by various workers ; these do not always lend themselves to an objective comparison. For these reasons, only the principal types of intercellular co-operations in immunogenesis will be recorded here. The first instance is afforded by the multipotential blood-progenitor or stem cells (of mesenchymal origin) which migrate into the analgen of various organs such as the foetal liver, thymus and the spleen at defined periods of intra-uterine life.60 The uncommitted, haematogenous stem cell interacts with inducers of lymphopoeisis to differentiate into either a T cell (thymus) or a B cell (bone marrow, bursa of Fabricius). A direct cell to cell interaction between lymphoid and thymus epithelial cells has been suggested but the possibility of a humoral factor, active at a distance, should also be kept open.55 In a second instance, cellular interactions influence the traffic patterns of these T and B cells in vivo. Thus, from their respective sites of origin, these T and B lymphocytes enter the circulation, migrate through the endothelial cells lining the venule, emerge finally inside the lymphoid organ (lymph node or spleen) where each seeks its own, specific microenvironment.35 Anatomically, within the ‘B area’, cells are observed to move from one such section to another.65 The last and the most defined step, cooperation between the T and B cells (along with macrophage) leads to antibody production. Experiments with phytomitogens and corticosteroids have further revealed the presence of sub-populations within these two major cell types.52 Although there is much argument with respect to the site, and the sequence in which these cellular types are activated, it is generally established that loss of one of these components leads to poor or no immune response.59 The manner in which the helper T cell amplifies the B cell response remains elusive.47 The possibility of an ‘inhibitor’ cell has also been entertained.21

In the concept of embryonic inductors, a determined group of cells acts upon an undetermined group by means of inductive substances or interactions between both tissues. The inducing substance (specific ?), from the inductor group, enters (how ?) the responding cell which controls the specific type of differentiation, much like a hormone. The environmental control of epithelial cells in vivo and in v i m has been demonstrated by the importance of epithelial-mesenchymal interactions in a variety of morphogenetic processes during amphibian and chick embryo development, during movements of cells in culture, association of foetal trophoblast and maternal decidua leading to LTH secretion in the mouse placenta (which does not proceed if either component is alone), during genesis and maintenance of thyroid characteristic in the mouse.32 Some salient features of this system are detailed below.

316

Kidney

Early differentiation in the ureter requires specific metanephric mesenchyme which, if left alone, remains undifferentiated without proper epithelium. Later, ureter provokes a specific nephrogenic reaction in specific meta- or mesonephric, or even lung or gastric, but not, intestinal, mesenchyme. Reciprocally, ureter is transformed into pulmonary, proventricular, or intestinal-like structures in the presence of lung, gastric or intestinal mesenchymes, respectively. Furthermore, association of metanephric mesenchyme with Wolffian duct results in differentiation into mesonephric mesenchyme; that of mesonephric mesenchyme with ureter into metanephric mesenchyme.90

Lung The primary inductor in pulmonary development is again mesenchyme. Chick lung epithelium in culture develops into bronchial patterns in the presence of mouse or chick lung mesenchyme. Pseudobronchi are obtained when lung epithelium is combined with proventricular, gizzard, intestinal or liver mesenchyme. Mesonephric, cephalic and allantoic mesenchymes have no noticeable effect on lung epithelium. The mesenchymal inductor can be filtered through vitelline membranes and appears to be a protein or a lipoprotein. Unlike the kidney, Dzffeerentiation 2, 1974

Cell-cell communications reciprocity seems to be lacking in this systern.26 Mesenchyme also controls the morphological and biochemical differentiation in the stomach.77 ‘This epithelio-mesenchymal interaction is proposed to proceed via specific collagen fibres.38 I n an in vitro analysis, it has been shown that mesenchyme is a major source of collagen associated with epithelial cell surface, that the presence of epithelial collagen is not dependent upon epithelial morphogenesis and that epilhelium influences the amount of collagen in mesenchyme.3x Does the mesenchyme only permit the development of epithelium or is its action truly instructive? Specificity of mesenchyme favours instructive capability.38 If a piece of mesenchyme from the branching bronchial regions is transplanted to morphogenetically quiescent trachea, one obtains the formation of supernumerary tracheal buds at the site of the graft.9 When a piece of mouse lung epithelium is combined with lung mesenchyme of the chick, the branching pattern of the explants was an intermediate of characteristics from the two species.82 The branching pattern of mouse mammary epithelia becomes salivary-like in combination with salivary mesenchyme. More important, when mammary or salivary epithelia were exposed to the simultaneous presence of both types of mesenchyme, their morphogenesis was typical of neither organ, but strongly inhibited, suggesting a mutual interference of morphogenetic influences exerted by either mesenchyme. Furthermore, regional specificity in the differentiation of chick skin derivatives resides within the mesoderm. Thus, the combination of prospective feather dermis with dermis of the foot, expected to form scales in situ, yields feathers ; prospective scale dermis forms scales even with epidermis from the back? Epidermis is also able to respond specifically to mesenchyme from organs other than the skin; 5 day chick epidermis forms a mucus-secreting, ciliated epithelium when combined with gizzard mesenchynie.56 Liver

Prehepatic endoderm must be successively subjected to the action of two mesenchymes, viz. cardiac and hepatic. The first inducer (cardiac mesenchyme) is necessary for the maturation of prehepatic into hepatic endoderm. This latter then differentiates into hepatic parenchyma in presence of specific hepatic mesenchyme. If this second reaction is allowed to progress in the presence of metanepric or mesenteric mesenDifferentiation 2, 1974

chyme in vituo, hepatic cords are formed that d o not synthesize glycogen. In the last stage of functional development, specific mesenchyme is essential where pseudohepatocytes, already formed in association with ‘non-specific’ mesenchymes, divide actively and synthesize glycogen.51 On the other hand, U DP-glucuronyltransferase7* or tyrosine transaminase74 activities attain ‘mature’ levels (and become cortisol-inducible) as soon as hepatocytes are removed from the embryonic environment. Conversely, one may study the influence of inhibitory embryonic environment on an autonomous hepatoma cell ! Components kept apart too long lose the ability to interact, and components separated following a sufficient period of interaction can develop even after separation As noted above, this sort of intercellular dialogue is reciprocal. In the absence of epithelium, the mesenchyme is organized into lacunae reminiscent of liver sinusoids. When epithelium migrates into such mesenchyme, the latter is transformed into endothelium which can undergo haematopoiesis. Thus, normal liver development calls for a reciprocal interaction between both the component cellular types leading to functional maturation of both.3*,51,74J* In conclusion, there is specificity in the range of tissues that can replace the normal component in inducing the developmental response. lnduction of mouse salivary epithelium is promoted only by ‘condensed’ mesenchyme-which may be obtained from parotid or salivary rudiment of the mouse or even in the salivary rudiment of the chick. Less specific are lung and thymus epithelia although they develop best in their own mesenchyme. Rather curiously, pancreatic epithelium is promoted even more strongly by salivary mesenchyme than by its own mesenchyme.Thus, three grades of epithelial behaviour may be distinguished: spreading with little or no cytodifferentiation (in absence of mesenchyme); formation of spheroidal, compact masses with ‘simple’ cytodifferentiation (in the presence of ‘common’ mesenchymal factors); and organized ramifications with ‘complex’ cytodifferentiation (in the presence of ‘specific’ mesenchymal factors). Reciprocally, various epithelia tend to aggregate mesenchymal cells in greater density in their immediate vicinity. It should be remarked that inductive interactions can occur across measurable distances (after separation by millipore membranes). Such interactions are also observed in annelids and molluscs where cell interactions between ectoderm and endoderm is indispensable for differentiation into shell gland.19 311

M. K. Agarwal Concluding speculations

The closed system of evolved and differentiated Eukaryotes is characterized by various types of specializations to assure the contact needed for homeostasis within and between the various component structures. In its simplest form, intercellular contact is assured by a variety of short range interactions (e.g. desmosomes, synapses, cytoplasmic bridges) that acquire the morphofunctional dimensions required for the cellular community in question. Cellular junctions with a much lower electrical resistance than cytoplasmic membranes are formed between animal cells in vivo and in tissue culture. Such junctions are particularly rich in embryonic tissues and seem to be uncoupled in certain malignant tumours. Biochemical studies in vitro, and on Lesch-Nyhan hybrids in vivo have further established the presence of intercellular interaction and exchange of required metabolites via long cytoplasmic extensions. Cell fusion experiments open up the possibility that the control and the influence of intercellular exchanges may be analysed in a defined manner. One step higher in organization are the long range interactions that proceed via very sophisticated junctions in nerve cells. In recent years, genetic analysis has made it possible to conclude that differentiation and functioning of the nervous system require contact between components far removed in the anatomical hierarchy of the adult brain. All evidence points to the presence of a chemotactic guidance system (nerve growth factors, neurotransmitters) whose disruption affects function in areas not directly affected by the mutation or the surgical lesion. An even more complicated level of cellular interaction is most clearly exemplified by the process of immunogenesis, where morphofunctionally distinct cellular entities participate successively towards the realization of a single phenomenon. These reticuloendothelial cells are derivatives of the embryonic mesoderm or mesenchyme. Their outstanding properties consist of liberal exchange between the free and the fixed compartments, transformation from one cellular type to the other, and rapid adjustment in the available volume depending upon the host requirement. They are popularly termed as the first line of defence and have been recorded as contributing to the maintenance of homeostasis in very diverse and varied forms of cellular phenomena. Apart from the structural ectoderm cornPonents, differentiated organs are comprised of 378

the tissue-specific epithelium or parenchyma of endodermal origin, and a somewhat undefined and uncared for mesenchymal compartment in the form of endothelial cells or the equivalent. Organogenesis tells us that specific mesenchymoepithelial interactions dictate the maturation of organ-specific epithelial self from the non-self both at the morphological and the biochemical level. In the most defined analysis, in this context, liver acquires gluconeogenic potential only if it is allowed to mature in the presence of organspecific mesenchymal factors. Should one assume that this mesenchymal something or another is switched off in the differentiated state? Recent studies on enzyme induction and on regeneration reveal that such mesenchymal influence may be recalled in the adult life. Intuitively, it is tempting to speculate that mesenchyme-derived reticuloendothelial cells may represent the surveillance and the co-ordination mechanisms in the host. In the long range, this might be the most important contribution of the RES cells in host homeostasis, beyond the immunogeneic potential which is unique to them. Scientific progress is dominated by fashion trends. Workers of the modern era are obsessed with the fascination of simplicity and ease of in vitro analyses. Such approaches are termed ‘molecular’ and ‘defined‘ even if they should be manifestations of transformation due to autonomy imposed by culture in isolated environments. Yet, autonomy remains the key feature of neoplasia which does not respect the compartmental barriers obeyed by the functionally integrated cells. If this were not the state of affairs, one could have cherished the hope of defining the manner in which endogenous cellular regulators (e.g. hormones), a variety of metabolic variants, and a whole range of noxious stimuli, may interact with a common internal receptor/ guidance system before being expressed as ‘response’ by cells possessing specialized functions. Finally, it is tempting to predict that, although the laboratory giants such as E. Coli and D . discoideum have a lot to tell us, the poor church mouse may also be endowed with regulatory mechanisms novel in biological hierarchy. ‘What is true of E. coli may also be true of an elephant’ at the molecular level, but an elephant is that much bigger to cope with in terms of control mechanisms that harness the molecular niche, Certain studies of the author were initiated in the laboratories of Dr L. J. Berry at Bryn Mawr. I should Differentiation 2, 1974

Cell-cell communications like to thank Drs F. Rosen (Roswell Park, Buffalo), I.B. Weinstein (Columbia, New York) and R. S. Snart (Shemeld, U.K.) in whose laboratories other aspects were variously developed. Still other studies were conducted as part of Equipe 106 of the C.N.R.S. Provoking discussions with Drs P. Feigelson (New York), B. S. Leung (Portland), F. Lundquist (Copenhagen), C. E. Sekeris (Marburg), and P. 0. Seglen (Oslo) are deeply appreciated. Space will not permit me to thank individually the numerous other colleagues, students and technical assistants who have contributed invaluably towards the completion

of these and other problems. Amid the professional excitement one often forgets the task of the nonscientific secretarial staff who collectively have to put up with trying and exacting demands of individual researchers; but it is to their p a h c e and understanding that I owe the final presenfation of this as of all other communications. M.K.A. is Charge de Recherche au C.N.R.S.

Received: April, 1974 Revised: June, 1974

References

1. Abercombie, M., National Cancer Inst. Monogr., 26, 249, 1967. 2. Agarwal, M. K., Sub-cell. Biochem., 1, 207, 1972. 3. Agarwal, M. K., Diferentiation, 1, 285, 1973. 4. Agarwal, M. K., Fed. Proc., 32, 538, 1973. 5. Aganval, M. K., Trans. Biochem. SOC., 1, 1185, 1973. 6. Agarwal, M. K., Biochemical Med. (in press). 7. Aganval, M. K., Differentiation, 2, 29, 1974. 8. Agarwal, M. K., Hoffman, W. W. & Rosen, F., Biochim. Biophys. Acta, 177, 250, 1969. 9. Alescio, T. & Cassini, A., J. Exptl. Zool., 150, 83, 1962. 10. Aronson, M., J. Exp. Med., 118, 1083, 1963. 11. Baker, J. B. & Humphreys, T., Proc. Natnl. 68, 2161, 1971. Acad. Sci. (US.), 12. Baylor, D. A. & Nicholls, J. G., Nature, Lond., 232, 268, 1971. 13. Benda, P. & Davidson, R. L., J. Cell. Physiol., 78, 209, 1971. 14. Bendicha, A,, Vizoso, A. D. & Harris, R. G., Proc. Natnl. Acad. Sci. (U.S.),57, 1029, 1967. 15. Bimbaum, G., Exp. Cell. Res., 56, 92, 1969. 16. Bourgoin, J. J., Cah. Med. Lyon, 49, 171, 1973. 17. Brachet, J., Proc. Roy. SOC.Lond., B., 178, 227, 1971. 18. Castro, J. E., Proc. Roy. SOC.,Lond., B, 185, 409, 1974. 19. Cather, J. N., Adv. Morphogenesis, 9, 67, 1971. 20. Chervenick, P. A. & Lo Buglio, A. F., Science, 178, 164, 1972. 21. Cohen, J. J., in: Cell Interactions, ed. Sylvestri, L. G., North Holland Publishing Co., 1971. 22. Crandall, M. A. & Brock, T. D., Science, 161,473, 1968. 23. Crick, F., Nature, Lond., 225, 420, 1970. Differentiation2, 1974

24. Curtis, A. S., J. Embryol. Exptl. Morphol., 23, 253, 1970. 25. Davidson, R. L., Fed. Proc., 30,926, 1971. 26. Daweron, F., Compt. Rend. Acad; Sci., 262, 1642, 1966. 27. Doerner, K., J. Theoretical Biol., 19, 275, 1968. 28. Ennis, H. L. & Sussman, M., J. Gen. Microbiol., 10, 110, 1958. 29. Ephrussi, B., Hybridization of Somatic Cells. Princeton University Press, N.J., 1972. 30. Ephrussi, B. & Weiss, M. C., Scientific Am., 220, 26, 1969. 31. Fawcett, D. W., Exptl. Cell Res. Suppl., 8, 174, 1961. 32. Fleischmajer, R. & Billingham, R. E. (eds.), Epithelial Mesenchymal Interactions,Williams and Wilkins, Baltimore, Md., 1968. 33. Furschpan, E. J. & Potter, D. D., Curr. Topics in Developmtl. Biol., 3, 95, 1968. 34. Garber, B. B. & Moscona, A. A., Dev. Biol., 27, 235, 1972. 35. Gesner, B. M. & Ginsberg, V., Proc. Natnl. Acad. Sci. (U.S.), 52, 750, 1964. 36. Giacobini, E., in: Cell Interactions, ed. Sylvestri, L. G., North Holland Publishing Co., 1971. 37. Godard, C., Pathol. Bid., 19, 175, 1971. 38. Grobstein, C., J. Natnl. Cancer Inst. Monogr., 26, 279, 1967. 39. Halpern, B., Pejsachowicz, B., Febvre, H. L. & Barski, G., Nature, Lond., 209, 157, 1966. 40. Harris, H., Proc. Roy. Suc., Lond., B, 179, 1, 1971. 41. Herron, W. L., Riegel, B. W., Myers, 0. E. & Rubin, M. L., Inv. Opthal., 8, 595, 1969. 42. Hidemann, W. H., Ann. Rev. Gen., 7 , 19, 1973. 43. Hill, M. & Spurna, V., Exptl; Cell Res., 50, 223, 1968. 379

M. K. Agarwal

44. Holley, R. W. & Kiernan, J. A., Proc. Natnl. Acad. Sci. (U.S.), 60,300, 1968. 45. Horridge, G. A. & Meinertzhagen, I. A., Proc. Roy. Soc., Lond., B, 175, 69, 1970. 46. Iwasaki, Y. & l'itot, H. C., L$e Sci., 10, 1071, 1971. 47. Jaffe, E. A., Nachman, R. L., Becker, C. G . & Minick, C. R., J. Clin. Inv., 52, 2745, 1973. 48. Katz, R., Nerve, Muscle and Synapse, Mc Graw Hill, N.Y., 1966. 49. Katz, D. H. & Benacerraf, R., Adv. Immurzal., 15, 1, 1972. 50. Kenney, F. T., Lee, K. L., Stiles, C. D. & Friti, J. E., Nature, New Biol., 246, 208, 1973. 51. LeDouarin, K.,Develop. Biol., 17, 101, 1968. 52. Levine, M. A. & Claman, H. N., Science, 167, 1515, 1970. 53. Loewenstein, W. R., Develop. Biol., 15, 503, 1967. 54. Loewenstein, W. R., Canadian Cancer ConJ, 8, 162, 1969. 55. Mandcl, T., Z. Zellfursch., 106, 498, 1970. 56. McLoughlin, C. E., J. Embryol. Exptl. Morphol., 9, 385, 1962. 57. Migeon, B. R., Kaloustian, V. M., Nyhan, W. L. & Young, W. J., Science, 160, 425, 1968. 58. Mintz, B., Fed. Proc-, 30, 935, 1971. 59. Mitchinson, N. A., Eur. J. Immunol., 1, 18, 1971. 60. Moore, M. A. S. & Owen, J. T., J. Exp. Med., 126, 715, 1967. 61. Moscona, A. A., Scientific Am., 205, 143, 1961. 62. Moscona, A. A., Proc. ll'atd. Acad. Sci. (U.S.), 49, 742, 1963. 63. Moscona, A. A., Develop. Biol., 18,250,1968. 64. Muir, A. R., J. Anat., 101, 239, 1967. 65. Nossal, G. J. V., in: Cell Interactions, ed. Sylvestri, L. G., North Holland Publishing Co., 1971. 66. Orr, C. W. & Roseman, S. J., Mem. Biol., 1, 125, 1969. 67. Pessac, B. & Defendi, V., Nature. New Biol., 238, 13, 1972. 68. Pitts, J. D., in: CIBA Foundation Symposium, ed. Wolstenhulme, G. E. W. & Knight, J.,

380

Livingstone, London, 1971. 69. Potter, D. I]., Furschpan, E. J. '& Lennox, E. S., Pror. Natnl. Acad. Sci. ( U S . ) , 55, 328, 1966. 70. Robert, L. & Robert, B., Path. Bid. Suppl., 21, 97, 1973. 71. Roelants, G., Curr. Topics in Microhiol. and Immunol., 59, 135, 1972. 72. Rubin, W., J. Cell. Biol., 52, 219, 1972. 73. Sengel, P., Ann. Sri. Nat. Zool., 20,431, 1958. 74. Sereni, P. & Sereni, L., Adv. Enz. Reg,, 8, 253, 1969. 75. Serrou, B., J. Med. Montpellier, 7, 281, 1972. 76. Sidman, R. L., in: Cell Interactions, ed. Sylvcstri, L. G . , North Holland Publishing Co., 1971. 77. Sigot, M., Compt. Rend. Acad. Sci., 254,2439, 1962. 78. Skea, B. R. & Nemeth, A. M., Proc. Natnl. Acad. Sci. ([J.S.), 64,795, 1969. 79. Stern, K., Ann. N. Y. Acad. Sci., 88 251, 1960. 80. Stocker, M. G. P., J. Cell. Sci., 2, 293, 1967. 81. Stossel, T. I'., New Eng. J. Med., 290, 717, 774, 833, 1974. 82. Taderara, J. V., Develop. Biol., 16,489, 1967. 83. Tomkins, G., Nature, New Biol., 239,9, 1972. 84. Trinkhaus, J. P., Lentz, J. P., Develop. B i d , 9, 115, 1964. 85. Uretsky, N. J. & Iverson, L. L., J. Neurochern., 17, 269, 1970. 86. Weiss, M. C., Todaro, G., Green, H., J. Cell. Physiol., 71, 105, 1968. 87. Weston, J. A., in: Cell Interactions, ed. Sylvestri, L. G., North Holland Publishing Co., 1971. 88. Whitcock, J. P., Cooper, H. L. & Gelboin, H. V., Science, 177, 618, 1972. 89. Wiener, J., Spiro, D. & Loewenstein, W. R., J. Cell. Biol., 22, 587, 1964. 90. Wolff, E., Curr. Topics in Develop. Biol., 3, 65, 1968. 91. Wolpert, G. & Gustafwn, T., Endeavour, 96, 85, 1967. 92. Wolstenholnie, G. E. W. & O'Connor, M., ed., Growth of the nervous system, (CIBA Foundation Symposium), J and A Churchill Ltd., London, 1968.

Differentiation2, 1974