Immune Cell Performances And Metabolism, an Ecological Interpretation

Pierre Sonigo
sonigo@cochin.inserm.fr
Génétique des Virus
ICGM-CNRS UPR415
22 rue Méchain
75014 PARIS, FRANCE

Posted on Heraclitean Biology Group web site June 23, 1999
Copyright 1999 by Pierre Sonigo

Abstract

The clonal selection of cells in the immune system is considered as a paradigmatic model of Darwinism at the cell level. However, it requires prior instruction of cells during embryogenesis and signals, concepts usually attached to instructive models. A more radical Darwinian postulate considers cells as selfish elements constrained directly by the nature and abundance of metabolic resources present in the antigen. As in ecological models, multicellular order in the immune system is thus driven by the structure of alimentary or metabolic networks. I examine here whether adaptation to metabolic resources at the cell level in the absence of cooperation is sufficient to explain immunological facts such as clonal selection, vaccination, self/non-self recognition, antigen presentation, cytokine network, glucocorticoid action and apoptosis.

" My investigations have shown me that in the blood of animals which have not been subjected to any treatment we must accept the presence of a number of normal bodies analogous to the "Antikorper," having their origin in the most widely diverse organs, and representing nothing more than nutritive side-chains, which in the course of the normal nutritive processes have been developed in excess and pushed off into the blood. " Paul Ehrlich (1)

Introduction : conceiving immunity without cell cooperation and signaling ?

In the clonal selection theory for antibody formation, it is considered that the signal initiated by the antigen causes selection amongst a population of cells carrying diverse molecules specialized in antigen capture. Interestingly, the antigen signal does not constitute an usable resource or a direct selection pressure at the cell level. To indicate this, and the absence of selective elimination of cells, the term of election might be preferred to selection (2). Although the clonal selection theory is considered as a paradigmatic example of Darwinism at the cell level, the signal concept is typical of instructive models. Indeed, signals require a previous learning process to be adequately produced by the emitting structure and precisely deciphered by the receiving one. Therefore, the final state must be virtually preexistent in the initial state, before the signal occurs, for example in the form of specific receptors able to recognize it. This is why signals are preformationist and problematic as regard to their evolutionary origin (3). In addition, understanding the evolution of signal exchanges in general is problematic : signalers, whatever their nature (multicellular or monocellular), are likely to emit wrong signals to optimize their benefits (see for example refs. 4-7 and references therein).

Models of global organization and regulation that emerge from selfish interactions and do not require cooperation between individual elements are not confronted with these difficulties. Such models exist in the fields of ecology or economy. We might thus question their universality, and especially their relevance to certain areas of cellular biology (8). However, in these models, it is necessary to define advantages (and associated selection pressures) at the individual level that differ from advantages obtained at the global level : for example, the primary selection pressure driving animals and plants in an ecosystem is not the global balance of the ecosystem, but the survival of the individual elements. In immunology, defense against infection is not defined at the individual cell level, but at the global organism level. Thus, to apply concepts derived from ecology in immunology, the selection pressures driving the immune response in the short-term at the cell level cannot result from the global immune function.

Clonal selection : cell competition for food

What kind of advantages could be obtained by selfish cells during the immune response ? It was proposed in the early ages of immunology, by Elie Metchnikoff or Paul Ehrlich, that immune functions derive from nutritive functions. Since then, multiple interactions between immunity and metabolism were studied, but their mechanisms remain elusive (see ref. 9 for examples and discussion). Such a difficulty might come from modern biology tending to classify the molecular world between two entities: one set of molecules is seen as involved in ubiquitous metabolic and house-keeping activities (for instance, metabolic substrates and enzymes driving metabolic chains); the other functionally distinct set would comprise those molecules involved in the regulation of specific cellular functions (transcriptions factors, kinases, antigens and signaling molecules, …). In contrast, in a Darwinian model of embryonic development, such a distinction is absent. Cell differentiation is not instructed by regulatory molecular signals. It is directly driven by metabolism, providing the selective constraints stabilizing random variations of cell phenotypes (10,11).

Is metabolic selection at the cell level able to provide a coherent interpretation of immune cell differentiation and function ? The simplest solution is that selection by signal would be replaced by selection by resources. Such resources would be of a metabolic nature, as present in the antigen. In the following story, let’s examine the effects of this translation : lymphocytes are depicted as diverse animals in a natural reserve, and antigens as diverse food. The story illustrates that clonal selection, leading to proliferation of the most efficient antigen-binding cells, might be interpreted as a competition between specialized predators for access to alimentary resources.

Mr Antigensignalling : The zoo director wants our research group to determine exactly which food is the best for each animal in the reserve. As food capture is a complex recognition and signaling system, we should take one individual of each animal species in the reserve for complete molecular dissection. Using detailed description of teeth, especially within frozen aliment-teeth complexes*, digestion mechanisms, 3D structural models and molecular design, we will surely determine how each specific food acts on the reproduction of each specific animal.

Mr Antigenfood : That is a great idea. It will convince the zoo director to buy a new powerful computer. So we can play the last flight simulator.

Mr Antigensignalling : Are you crazy ? We have a huge repertoire of billions of rapidly reproducing and evolving animals in the reserve. With such a diversity, we will have huge calculation needs to reconstitute every molecular details.

Mr Antigenfood : Don’t worry, Sig. The experiment is already ongoing without the computer. First, I have asked to reduce the overall feeding in the reserve. Then, every week, we provide only one kind of food. The animals will compete for the food. At the end of the week, we just count the animals. The animals who have the best capture and digestion efficacy for the food will be for sure the most activated and reproductive in the reserve. The less efficient animals have no access to the food. They just sleep and are not able to reproduce.

Mr Antigensignal : Good idea ! And to make the animal reproduction and activity more detectable, why don’t we use food adjuvants ? Let’s attach the food to a big energetic molecule. **

* refer to crystals of antigen-antibody complexes.

** Immune response is more efficient when the antigen is associated to a molecule called adjuvant. In an extreme situation, small molecules (haptens) are not able to induce an immune response if they are not coupled to a large protein (carrier) (see ref. 12 for a review). As the hapten is able to interact specifically with the specialized antigen receptor, it remains at present unclear within the signal-triggering representation why it is not able to cause cell proliferation and activation when alone. This might be more easily understood if lymphocyte proliferation does not require a signal, but a metabolic advantage provided by the antigen as a nutritive resource. The carrier molecule might thus constitute a metabolic resource absent from the hapten.

Vaccination and memory : oscillations in prey-predators relationships

Vaccination relies on the amplification of specific lymphocyte clones by an immunogen resembling the micro-organism in an innocuous form. Such an amplification is the basis of immunological memory. Boosts are necessary to maintain the immunity on the long term. If an antigen is considered as a metabolic resource, as proposed above, immunological memory might result from non-synchronized prey/predator oscillations following a Lotka-Volterra model (for examples and recent references, see the special issue of Science ref. 8). In such a situation, to explain vaccination mechanisms, there is no need for self/non-self recognition or cooperation between cells. Such a hypothesis is illustrated in the next fable, where it is shown that usual vaccination techniques (attenuated vaccine) can be used to vaccinate a river against red fish infestation.

Mr Clearwater (director of the River Health Institute): The river suffers regularly from a strange disease called redness : it is a red fish proliferation with multiple pathogenic consequences. Due to rainwater overflow the fishes are transmitted from a healthy reservoir where they surprisingly caused no anomaly. When the fishes infest the river, they eat algae and insects that purify the water. With less algae and insects, the water is different and other animals and plants in the river suffer from it. We have to eradicate the fishes from the river.

Mr Drugdesign (a cousin of Mr Antigensignalling) : let’s design an antifishotic. To avoid toxicity for other non pathogenic fishes in the river, we just have to know the exact molecular structure of the fish mouth, and we can design a poisoned insect that will specifically fit the mouth of the virulent fish, so the other fishes will not eat it.

Mr Vaccine : You will poison the whole river ! Bigger fishes will also eat the poisoned insects, whatever their structure ! I have a better idea. Let’s breed a huge amount of red fishes in our experimental aquarium. We will anesthetize them slightly, so they will not cause any damage, before injecting them massively into the river.

Mr Drugdesign : Are you crazy ? Injecting attenuated fishes in the river ! How do you expect this to work ?

Mr Vaccine : That’s easy. All the kingfisher birds in the neighborhood will come and multiply above their usual number this summer given the abundance and easy capture of their favourite resource. Next year, the greater number birds will easily control the red fish proliferation.

Mr Drugdesign : I am afraid that you will need to inject fishes regularly to maintain a sufficient number of kingfishers. We will not waste our time and money breeding red fishes to feed these birds.

Mr Vaccine : Given the long life expectancy of the birds, I think an anesthetized red fish boost every ten years will do the job.

Self or non-self, (as well as antigen) are central concepts in immunology. However, their initial definitions, derived from observations of graft rejections or infections, suffered from numerous exceptions (i.e. autoimmunity or tolerance) the mechanisms of which still remain debated (see for example refs. 13-16). I suggest the following adjustments of previous definitions :

Self : elements participating in the metabolic exchanges set up during the previous history of the cells (including embryonic development).

Antigen, or non self : a resource capable of disturbing the dynamics of the metabolic network previously established between cells in the immune system.

The following fable, inspired again by ecological models, attempts to illustrate these definitions. The prairie with only three components is used as a naive image of a non linear system that auto-organize by dissipating a flux of energy (food) (17). Possible analogies between this simplistic ecosystem and the cellular world are commented in the legend.

A very little bird made droppings in the desert. The droppings allowed the growth of a blade of grass. The grass fed ants. In turn, the ants fed the bird that laid more droppings. More droppings made more grass that made more ants, that fed more birds. The droppings were the egg for a wonderfully organized prairie, composed of three specialized and interdependent metabolic organs : grass, ants, and little birds.*

It was well balanced for years. Although the inhabitants were eating each other, the prairie was a model of harmony, and no one was able to outgrow the others.**

Then a cockroach came, which started to eat the ants. Less ants fed less birds that made less droppings, that made less grass : the whole prairie was ill. Disease spread all over, except for the happy cockroaches and for a family of bigger birds. These red wing birds came because they were able to eat the cockroaches : they multiplied and their secretions fed many newcomer animals, setting up a new organization in the prairie. However, the big birds were so voracious that they ate all the cockroaches. No more cockroaches to eat. They and their followers had to leave after having cured the prairie ***.

Later came a slow little cockroach. It was not eating enough ants to disturb the organization and life cycles in the prairie. It was not reproducing quickly enough to satisfy the appetite of the red wing bird and support its reproduction. It stayed in the prairie for years ****.

* self/non-self : All organs of the prairie (birds, ants, grass), that are involved in the established dynamics of food exchange constitute the " self ". In such an ecosystem, the " self " is not the result of typical cooperation based on learning and signaling. It results from stable prey-predator or alimentary chain relationships. It might be the same between cells in a multicellular organism, for instance in the immune system.

** cancer : Individual cells of the organism, like elements of the ecosystem, are selfish. However, they are highly interdependent because the growth of one component requires a parallel growth of the others. In biology, a coordinated growth of multiple cellular components is typical of embryonic development, whereas unbalanced growth is typical of cancer. In the above example, a local imbalance of resource distribution allowing a growth advantage of one component of the prairie is the equivalent of a benign tumor. For unlimited growth of one element to occur (cancer), the rapidly multiplying element (or cell) must loose its food dependence from the other components (or cells) : this would require a maintained anomaly of resource distribution and loss of previous food specialization. Indeed, to grow independently from other cell types, cancer cells must acquire a capacity to produce the whole alimentary chain themselves. This is supported by the loss of differentiation/specialization of cancer cells and the requirement for redistribution of metabolic supply, for example through neoangiogenesis (18).

aging : the above prairie will grow to a maximum size, for example because of diffusion limits or exhaustion of the soil. Then, it will possibly stabilize and decline : less grass feeds less ants that feeds less birds, etc. Such a decline might corresponds to an aging process in the cellular world. During the decline, anomalies of resource distribution will be more frequent explaining the increased frequency of local uncoordinated growth. Similarly, cancer frequency increases with age.

*** acute infection : The first cockroach corresponds to a strong antigen because it diverts sufficient resources to displace the ecosystem equilibrium and to support the growth of new predators. This underlines the importance of the amount of antigen in controlling immune response efficacy (see below). In an ecosystem, the multiplication of new predators may initiate a new alimentary network. In an analogous manner, activated macrophages capturing the antigen initiate a new chain of cellular proliferations.

**** chronic infection : The " slow little cockroach " does not belong to the initial elements. However, it does not modify the resource equilibrium enough to induce its own elimination and persist. It might have deleterious consequences only in the long term (persistence, AIDS). In the short term, it might be considered as an element of the self.

Advantages and requirements of specialized predation : antigen presentation in the immune system

How do the proposed definitions of self and non-self accommodate experimental observations and knowledge concerning T-cell molecular mechanics ? T-cells are MHC restricted : they recognize antigens only when processed by proteolysis and presented in the context of an homologous MHC molecule. T-cell receptors are generated by random genetic recombination. Thymus cells carry MHC molecules loaded with diverse peptides from the organism. During embryogenesis, three fates are observed : T-cells that do not react with MHC die. T-cells that react with a high affinity to MHC also die (see a later paragraph for discussion about mechanisms of cell death). Only T-cells that react with an intermediate affinity are positively selected to constitute the T-cell repertoire (reviewed and discussed in refs. 13-16).

Let us examine the consequences of the alimentary chain hypothesis, which considers that peptides presented in the MHC constitute an essential food supply for the T-cells. This easily explains death of low affinity cells because they are unable to get peptide food from the MHC and starve. Death of cells with a high affinity receptor are more difficult to explain. High affinity might cause exhaustion of the peptide preys by their T-cells predators, and rapid subsequent disappearance of such predators. It might also cause " mechanical " difficulties in capture and internalization of presented peptides. Finally, the cells with the intermediate affinity will engage in a dynamic equilibrium of food exchange, based on peptides from the self. In the normal situation, the number of T-cells is stable and adapted to the abundance of "self-peptide food" provided by normal cells. This is supported by the presence of autoimmune reactive clones in the normal situation.

Destructive auto-reactivity occurs by displacement of this equilibrium to the benefit of T-cells. In the prevalent regulation or signal-based interpretations, auto-immunity is considered as a dysregulation of T-cell reactivity, resulting for example from inadequate signaling  (14,15). This explains why autoimmunity might follow cellular destructions of diverse cause (traumatic, infectious, etc...). In the metabolic-based interpretation, self peptides constitute an important metabolic resource for T-cells. The consequences are similar : autoimmunity might also result from a transient increase in self peptide production, increasing in turn the number of self consuming T-cells.

When an external resource (potential antigen) is introduced in the system, the succession of events will depend on the amount and diversity of the new molecules. If the amount is low, or the diversity is high, specialization for capture will not be a sufficient selective advantage and all the resources will be captured by non specialized cells (phagocytes). If the amount of a homogeneous resource is high enough to support specialization for it, specific immunity will develop based on selected B and T lymphocytes. This is illustrated by the low efficacy of the specific immune response despite an intense macrophagic activation, when confronted to highly variable pathogens such as Human Immunodeficiency Virus.

Interestingly, T-cells do not specialize for capture of the antigen directly. They recognize peptide products of antigen digestion by the phagocytes and antigen presenting cells. As structural diversity of short peptides is lower than structural diversity of larger molecules, the repertoire of antigen receptors (either immunoglobulins or T-cell receptors) will cover more easily a repertoire of short peptides (either linear epitopes or processed antigen, respectively) than a repertoire of large molecules. Processing thus allows a smaller number of cells to cover the whole diversity of possible resources. Direct specialization for large molecules (peptides, glycolipids) will be possible only when they contain structural repetition, as it is the case for example at the surface of infectious agents or for T-independent B antigens (19).

Cytokines : a food network

Cytokines (from cyto = cell and kine = movement) are proteins secreted primarily from leukocytes that bind to receptors on the cell surface, with the primary result of activating cell proliferation and/or differentiation. Cytokines are remarkable for their ubiquitous and redundant spectrum of activities, as specificity for a given cell type is exceptional. For these reasons, the " cytokine network " is often referred to as a typical example of complex regulated exchange of information between cells, necessary for immune response coordination (see refs. 21,21 for review). It is remarkable that the cytokine network has many features of an alimentary network : specialization without strict specificity, importance for activation and reproduction, succession of local exchanges in a micro-environment. I hypothesize that cytokines constitute a proteic resource for cells, explaining their trophic activity. I called it " cytokine-steak " hypothesis : given the weight ratios, an active dose of cytokine for a cell is equivalent to a nice piece of meat for us ! This food network might have originated as a consequence of the metabolic importance of proteins and amino-acids. Accordingly, cytokines are highly related to amino acid metabolism (22) or nutrition balance (23,24).

Would alimentary specialization at the cell level be sufficient to explain the diversity and functions of cytokines and cytokine receptors ? Some answers might come from comparison of animal cells with multicellular animals. Although amino-acids are essential building blocks, their synthesis is unequally distributed amongst the living world. It requires for example nitrogen fixation, a central element in ecosystem formation (25,26). To a greater extent than glucids or lipids, amino-acid and proteins often constitute a limiting resource in the animal kingdom. Animals obtain amino-acids from other organisms rather than by de novo synthesis. In this case, amino-acids will not be captured as free molecules, but included into proteins. Although life uses a limited number of amino-acids, diversity of proteins is susceptible to generate a large variety of food specialization. Animals are indeed adapted for efficient capture of the most abundant protein food in their proximity. For example, some are specialized for vegetal proteins capture, other for animal protein capture. It seems that animals are entirely built around this specialization : teeth, digestion apparatus, locomotion apparatus, metabolic equipment often reflect protein specialization (vegetarian, carnivorous ...).

In the same way, cells might be adapted to the most abundant protein resource in their proximity, as reflected by the specialization of their surface receptors. Just like animals, cells are limited by an hydrophobic " skin " (lipid membranes) which represents a barrier to hydrophilic substances. Cells had to develop a dynamic system of protein capture using a wide ensemble of " sticky " proteins located at the membrane. These membrane molecules, specialized in binding and internalization of aliments, especially proteins, are exemplified by members of the immunoglobulin superfamily (27). This superfamily is one of the most diverse and abundant protein family. It includes for example adhesion molecules (see also below for adhesion and apoptosis), and cytokine or olfactory receptors. Interestingly, some receptors are related to amino-acid transport proteins (for a recent review, see ref. 28). Finally, what we call a cytokine receptor might be considered as a specialized tool for capture of proteins from the cellular microenvironment. If proteins constitute a limiting resource in the cellular world, like in the macroscopic world, successful capture will condition cellular activity. Would cells be more complex than multicellular animals ?

Neurology and immunology : the roots of thought

Many authors have underlined analogies between the immune system and the nervous system. Whereas the immune system is composed of mobile cells and involve local exchanges through membrane receptors, the nervous system is composed of fixed cells and distant exchanges occur through neural fibers and synapses. During development for example, neurons compete for proteins like Nerve Growth Factor (NGF), located at distant sites. The amount of NGF determines precisely the amount of neural cells that will survive and innerve the NGF-secreting cells (29). In both the immunological and nervous system, in addition to cytokines and growth factors, amino acids and substances involved in amino-acid metabolism also have important biological activities : this is the case for example with tryptophane, that possesses neurological and immunomodulatory properties (30), with neurotransmitters and synthetic drugs, that are closely connected to amino acid metabolism, or even with Nitric Oxide, an essential element of nitrogen metabolism (31). Metabolic fluxes analogous to those proposed in the cytokine network might constitute the organizing forces of the nervous system. Selective stabilization of synapses (32), or memory (see the paragraph about vaccination), might result from the establishment of efficient " alimentary " cycles between cells of the nervous system. The next image illustrates the parallels between a sensitive function and a nutritive function.

In a tree, the aspect of the leaves reflects the nature of the resources found at the tip of the roots. The same is true with neural roots (axons and dendrites) and neural leaves (neurons) : the sensitive neuron reflects what happens at the tip of its roots. We wonder whether the tree is happy when water and nutrients are abundant at the roots. Perhaps the tree is in pain when the roots are dry ?

Glucocorticoids : feeding the foetus and the immune cells

Glucocorticoids are amongst the most potent anti-inflammatory molecules. They are exemplified by cortisol, a natural hormone secreted for instance during pregnancy and effort. Cortisol itself is related to cholesterol, a central element of lipid metabolism. Metabolic effects of glucocorticoids are multiple: hyperglycaemia through stimulation of neoglucogenesis and decreased glucose uptake and use in peripheral tissues, increased lipolysis and free fatty acid release, osteoporosis through phosphate and calcium redistribution, muscle catabolism and protein redistribution. They also have dramatic anti-inflammatory effects, whose mechanisms are still subject of intense research : indeed, whereas glucocorticoids receptors appeared in the 1980s as paradigmatic sequence-specific ligand regulated transcription factors, recent data question whether DNA binding is required at all for many of the physiological functions of the glucocorticoid receptors (33,34).

I propose that metabolic and immunological effects of glucocorticoids represent two sides of the same coin. By causing liberation of stored metabolic resources (glucose, amino-acids, calcium...), which is physiologically essential during pregnancy for example, these steroids might be able to satisfy the metabolic needs of immune cells, without requirement for inflammatory attacks and protein capture specialization. Another short fable illustrates these ideas : inflammation is depicted as auto amplified and destructive fights for food in a forest. These inflammatory focuses are relieved by the dramatic " hormonal " action of water. Water in the forest, like cortisol in the organism, is able to increase the global amount of immediately available nutritive resources.

It was dry for a long time, and food was lacking in the forest. Animals were starving. Any dead plant or animal induced the formation of violent gatherings where everybody fought for the cadaver. The fight for the first cadaver generated in turn more deaths and cadavers generated more fights. Everybody started to eat everybody in these gatherings, leading to the destruction of essential elements of the forest. Then came the rain season. Herbs and fruits appeared, and all the fights suddenly stopped.

Apoptosis : starvation and death

Apoptosis, also known as programmed cell death, is one of the most fascinating phenomenon in cell biology (35). It is implicated in many physiological or pathological processes : development, immunology, cancer, cardiovascular diseases, etc. It corresponds to an active process of cell death, differing by many morphological and biochemical features from other mechanisms of cell death. For example, it differs from necrosis due to mechanical injury. In contrast to necrosis, apoptosis requires protein synthesis, and the activation of elements of the mitotic machinery. It is accompanied by intense protease activation (caspases), mitochondrial modifications, tissue acidosis, and has different immunological consequences than necrosis (ref. 36 and references therein). Apoptosis has been associated to diverse ligand-receptor systems, with positive or negative regulatory effects, which are still a matter of controversy. To explain the complexity and opposing effects of regulatory signaling in apoptosis, it has been proposed that apoptosis might be the consequence of incomplete or unbalanced signaling (see ref. 37 for review).

I have also tested the possible explanatory value of changing the concept of signal to that of resources in the interpretation of experimental observations in the field of apoptosis. The typical example of apoptosis during embryonic development concerns the formation of interdigit spaces (38). Apoptosis occurs after formation of five vascular axes in the cellular mass forming the hand bud. Cells located around the vessels survive and form the fingers. Cells at a distance from vessels will undergo apoptosis. Given the known limits of diffusion of metabolites and gas from the blood vessels, this might be easily viewed as metabolically determined. Another example concerns epithelial cells that multiply actively when rooted in the basal membrane, which is the equivalent of a nutritive soil for the epithelium, and die in apoptosis when " homeless ", ie when their attachment through adhesion molecules is inhibited (39). I suggest that " homeless " cells die just like uprooted trees. As already mentioned, in the maturation of the immune system in the thymus, T-lymphocytes, the receptors of which show no or too high an affinity for surface proteins of other cells, also undergo apoptosis (40).

It is likely that deficiencies and imbalance in metabolic resources will have deleterious consequences in the cell. In usual interpretations, apoptosis is caused by the engagement in a specific program, in response to external signals (41). The evolutionary advantages provided by death signals at the cell level remain elusive. This problem might be solved by considering that metabolic problems, rather than signaling, are the primary cause of apoptosis. Indeed, predictable consequences of metabolic starvation are compatible with the pattern of modifications associated with apoptosis, which might reflect attempts of compensatory metabolic adaptation. Lack of amino acids for example will be revealed more dramatically when protein synthesis is active, explaining the association of apoptosis to protein synthesis. It might be accompanied by an increase in protease activity, necessary for freeing required amino acid from protein stocks. Metabolic imbalance is likely as well to induce mitochondrial modifications, and tissue acidosis given the central role of this organite in providing chemical energy to the cell. Variations in diverse protein kinase activities might result from metabolic problems, rather than cause them, as a consequence of reduction of the ATP and nucleotide triphosphate, main substrates of kinase activity.

Discussion : signal versus resource?

The distinction between a signal and a resource lies at the very heart of the present discussion. In immunology, ecological models considering that lymphocytes compete for antigen as a resource have been proposed (ref. 42 and references therein). It demonstrated the relevance and usefulness of ecological models in this context. However in these studies, the resource is equivalent to the signal (survival signaling) and is not of a metabolic nature. Ecology usually distinguishes food, non-food resources (sunlight, rain, shelter, etc.), and informational cues (length of day, pheromones, scents, etc.). What about the equivalent classes at the cell level ?

One basis for signal/resource distinction could be of a kinetic nature: a molecule would be said to convey a signal when causing a biological effect before its chemical energy is actually used; as an example, ligand-receptor interaction induces kinase activities before the ligand is internalized and metabolized to produce ATP. Within that definition, however, the signal function is associated to the resource function and is conveyed by the same molecule. Similarly, food induces saliva secretion before being actually digested.

It might be also objected that the signaling function of molecules is demonstrated by the capacity of " cheating ", as usually realized for therapeutic purposes (see for example ref. 43). This is due to the fact that in cells, any molecular resource is subjected to a chain of successive treatments : binding, internalization, enzymatic activation, metabolic steps... Any molecule susceptible to use the initial steps of the pathway, and to escape the late steps can be considered as a signal. Such dissociated properties for a given chain are widely exploited in pharmacology. Dissociated properties for one chain replace the classical requirement of two classes of chains : signal cascades and metabolic chains. Finally, as the end of a metabolic chain is often the beginning of another, in a more general formulation, a molecular signal may correspond to any element of a metabolic network.

Other considerations for a signal derive from concentration. Indeed, what we usually define as a molecular signal should be active at a very low concentration : 100-500 nM for example in the case of a hormone like cortisol. A few picomoles of some cytokines are sufficient to produce a cell response. Food or resource is thought of at much higher concentrations. Amino acids concentrations are so in the range of 50-500 uM. Typical metabolic resources such as pyruvate, lactate, and glucose, are present in millimolar concentrations. Yet, if the organism is an auto-organized non-linear system where the dissipation of energy corresponds to metabolic reactions chains, any molecule able to modify the efficacy of a reaction may non stochiometrically affect the order of the system. In such a system there will be no dose-effect relationship. On the contrary, a low concentration will define a rare molecule and the rarity may increase its value and effect as a resource for the system. This is the case with vitamins, exemplifying the limitation to distinguish between signal and resource on a quantitative basis.

The location of the resource in a hierarchical metabolic network, and its relative variations, might be more important than its initial concentration. Biological effects will be more pronounced for molecules acting at the lowest hierarchy of the metabolic tree (simple compounds : NO, calcium, ATP or cAMP, simple lipid, free aminoacids) than for molecules acting at the highest branches and requiring specialization for capture (composed compounds : steroids, insulin, cytokines, antigens, etc...). Composed compounds will act only on cell populations specialized for their capture : whereas a cytokine will reorganize a large population of lymphocytes, an antigen will act only on a few clones. Similarly, in a hierarchical food network, primary producers or primary resources variations might induce more extensive reorganization than variations of final resources used by specialized top predators. Light or rain variations will have more effects on the ecosystem than French fries.

Finally, at the cellular level, all molecules, whatever their denomination as minerals, food, signal, hormones, mediators, antigens, etc., are functionally "symmetrical" : all of them participate in the organizing metabolic network and all are susceptible to modify the dynamic equilibrium and thus the structure of the system. I hope that the definitions and descriptions proposed here will stimulate studies based on exchange of concepts and mathematical tools between ecology and cellular biology.

Acknowledgments

This text is derived from discussions within the Heraclitean Biology Group (http://www.heraclitean.com//). Many thanks to Jacques Leibowitch, Sergei Atamas, Jean-Jacques Kupiec and Robert Naviaux for their invaluable help, to Geneviève Milon, Isabelle Tardieux and Penny Starfield for corrections of the manuscript. I also thank D. Forsdyke for providing P. Ehrlich article on-line (http://post.queensu.ca/~forsdyke/homepage.htm).

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Most articles are cited for general concepts and reviews rather than original observations or specialized facts. When possible, I have tried to select the most recent and up to date references.

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