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--> 8 Cytokines and Nutritional Status: Possible Correlations and Investigations Jeffrey L. Rossio1 Introduction What Are the Cytokines? The cytokines are a relatively diverse group of low molecular weight (8,000–30,000 daltons) proteins that act to transmit information among the cells involved in immunological responses. On one level, they can be thought of as the "hormones" of the immune system. However, as will be explained shortly, the cytokines can have many important roles including cell activation, effector function (for example, killing of virus-infected cells), immune suppression, and induction of cell differentiation. The main function of the cytokines is to promote, sustain, and terminate an immunological response that is appropriate for the pathogen or other antigen toward which the response is directed. This chapter will give an overview of the cytokine system and the variables to be 1 Jeffrey L. Rossio, AIDS Vaccine Program, National Cancer Institute-Frederick, Cancer Research and Development Center, Frederick, MD 21702 The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
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--> considered when studying the relationships that may exist between the cytokines and nutritional status. The Nature of the Cytokines Biochemistry Almost all cytokines are glycoproteins secreted from a variety of cell types (Casciari et al., 1996). Recombinant cytokines have been expressed in bacterial systems where glycosylation does not occur and seem to have equivalent biological activity, at least vitro. However, the state of glycosylation may have some effect on the stability of cytokines in the circulation, and perhaps on biological half-life. Most cytokines work as monomers, although some exist as homodimers (for example, interleukin [IL]-10), heterodimers (for example, IL-12), and trimers (for example, tumor necrosis factor-alpha [TNF-α]). Mode of Action The cytokines act by binding specifically to receptors on the surface of target cells. Most cytokine receptors have been well characterized, and many have been isolated, sequenced, and cloned. Some receptors belong to the immunoglobulin superfamily, that is, they consist of glycoproteins with characteristic intra- and interchain disulfide bonds forming looped structures. Other cytokines use receptors in the 7-transmembrane family, which is known to work through the activation of intracellular G-proteins. Receptor structures vary from single chains to multimers of two or three chains. Also, some receptor peptide chains are shared among receptors for more than one cytokine. In some cases the receptors may be present (at low levels) on resting target cells; when these cells are activated during immune responses, the receptors increase in number. In other cases, receptors appear de novo following activation. This is an important characteristic of cytokine regulation. Principles of Cytokine Action Janis Kuby, in her textbook, Immunology (1994), notes three basic principles that must be taken into account when studying cytokines. First, the cytokine molecules tend to be very pleiotropic. That is, each one has numerous biological functions. At first, the cytokines were described in terms of their functional activities, for example "T-cell growth factor" (now, IL-2). However, when the cytokines were isolated and their amino acid sequences were determined, it was found that many molecules with diverse biological activities were, in fact, the same. So for instance, the molecule called interleukin-1 or IL-1 has at least 50 unique, separate names related to unique, separate biological
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--> activities. The "interleukin" nomenclature was, in part, developed to unify all the various names for the cytokines into a coherent system. A second important principle regarding cytokines is that they are redundant. That is, several unique cytokine molecules have the same biological function. One example is the ability of some cytokines to act as an endogenous pyrogen, to induce fevers. This was a property originally ascribed to IL-1, but later, at least two other cytokines, IL-6 and TNF-α, were shown to have similar activity. This feature makes sense in terms of natural processes, since redundancy ensures that if a problem develops in the production of a single cytokine, the entire immunological process will not become ineffective. However, the redundant nature of cytokines often poses problems in measurement of single cytokines using biological assays. It is important to realize that a single biological effect might be due to several different cytokines. A third characteristic of cytokine biology is that the cytokines are often synergistic. A single cytokine molecule may not have a strong biological effect, but when it is combined with other cytokines, the effect emerges. It is now known that the cytokines often work in cascading pathways, with one cytokine inducing the production of one or more cytokines so that the effect becomes amplified. Cytokine Experimentation Quite a bit is known about the cytokines and how they control immunological reactions. The cytokine system is quite similar in all vertebrates, and models developed in mice and other laboratory animals have been used extensively to dissect the roles of the various regulatory factors. In recent years, the techniques of molecular biology and genetics have allowed the development of transgenic mice that overproduce individual cytokines, as well as gene knockout animals that are unable to produce specific cytokines. The study of cytokine biology is complicated somewhat by the regulation imposed on in vivo cytokine activity. Cytokines are induced during the course of immunological responses, and their measured levels can correlate with the extent and nature of these responses. However, as discussed earlier, the cytokines work by binding to cell surface receptors, and these also are regulated during immunological responses. So, it is necessary to measure not only cytokine production but also the ability of an individual to respond in a given circumstance as a result of appropriate receptor display. Other authors in this volume have noted that during periods of high stress, such as those that occur during Ranger training, a substantial proportion of cells from the trainees had lost many surface markers. If the cytokine receptors are among these markers that are lost, this could explain the lack of proper cytokine regulation of an immune response and increased disease susceptibility in this population. This possibility has not been studied and would be a fruitful area of investigation.
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--> Classification of the Cytokines Since cytokine biology is so complicated, it is a good idea to try to compartmentalize the various cytokines by function. Joost Oppenheim of the National Cancer Institute's Frederick Cancer Research and Development Center, who is a codiscoverer of two of the cytokines (IL-1 and IL-8), and this author have suggested such a classification scheme, which may make it easier for a nonexpert to understand what to look at when studying cytokine actions (Oppenheim et al., 1993). An outline of the scheme is presented in Table 8-1. Inflammatory Cytokines The first group, and one of the most important, includes cytokines that are important in the development of inflammatory responses. There have been many studies of these activities because of the desire to control inflammation. Among the cytokines that fall in this category are IL-1, TNF-α, interferon-gamma (INF-γ), IL-8, and IL-17. T- and B-Lymphocyte Growth Factors This group is large and includes many cytokines that control the growth and differentiation of T- and B-lymphocytes, the cells that recognize foreign materials, initiate immunological responses, regulate those responses, and conduct many of the immunological effector activities (cell killing, antibody production, etc.). Cytokines in this group are the T-cell growth enhancers IL-1α, IL-1β, IL-2, IL-4, IL-7, IL-9, and IL-15 and the B-cell growth factors IL-4, IL-6, and IL-14. Note that some cytokines appear in more than one category, which emphasizes the pleiotropic nature of these molecules. TABLE 8-1 Classification of the Cytokines Inflammatory cytokines T- and B-lymphocyte growth factors Hematopoietic growth and differentiation factors Chemotactic agents Inhibitory/regulatory factors Cytotoxic/cytostatic inducers and effectors Other factors
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--> A distinction is sometimes made, which has been mentioned by other authors, between cytokines in this group that primarily enhance cell-mediated immune responses (Th1 responses) versus those that promote antibody or B-cell responses (Th2 responses). In mice, it appears that two different populations of helper T-cells produce different profiles of cytokines, but the situation is less clear in humans. At any rate, the Th1 group of cytokines includes IL-2 and IFN-γ, while the Th2 group includes IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Hematopoietic Growth and Differentiation Factors The first cytokines licensed for clinical use fall in the category of hematopoietic growth and differentiation. These molecules encourage the production of new white blood cells (WBC) from precursor cells in the bone marrow. Whenever WBC are consumed or killed (inflammatory reactions, blood loss, AIDS, chemotherapeutic insult, radiation exposure, etc.), these cytokines can be administered to hasten the recovery of normal cell numbers. Factors to enhance recovery include GM-CSF (granulocyte-macrophage colonystimulating factor), G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage/monocyte colony-stimulating factor), SCF (stem cell [activating] factor), IL-3, IL-5, and IL-11. Chemotactic Agents A growing family of cytokines acts to attract various types of cells to the site of nascent or ongoing immune or inflammatory responses. These "chemokines" are the newest group of cytokines and include IL-8, RANTES2, MIP (macrophage inflammatory protein)-1α, and MIP-1β. Inhibitory/Regulatory Factors Several cytokines are important in the control of immune responses by limiting the extent of response, for example, by terminating a reaction when the inducing substance has been removed. These include IL-10; IL-13; IFN-α, -β, and -γ; and transforming growth factor (TGF)-β. Cytotoxic/Cytostatic Inducers and Effectors Some cytokines have direct or indirect effects resulting in the killing of cells bearing foreign antigens, such as viral-infected cells, cells infected with intracellular parasites, or cancer cells. These include IL-12, TNF-α, and TNF-β. 2 RANTES: Regulated on Activation, Normal T-Cell-Expressed and -Secreted; a chemoattractant and activating factor for T-lymphocytes (mononuclear leukocytes).
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--> Other Growth Factors The above list is not exhaustive; many other cytokines have been described. Also, the list keeps growing as new factors are described, isolated, and characterized (Aggarwal and Puri, 1995; Callard and Gearing, 1994). Measurement of Cytokines Fortunately, the measurement of cytokines is becoming routine in the research laboratory, thanks to the development of sensitive and specific enzymelinked immunosorbent assays (ELISAs) (Kopp and Holmlund, 1996; Whiteside, 1994). In the past, reliance on assays for the biological activity of cytokines made measurement much more difficult, partly due to the inherent variability of biological systems and partly due to the pleiotropic and redundant activities of the cytokines themselves (Coligan et al., 1994). Several manufacturers offer ELISA capture assay3 kits with all the required reagents and standards necessary to evaluate cytokines in serum or plasma. In addition, several sources sell matched antibody pairs in bulk, including a capture antibody and a biotinylated secondary antibody. With these reagents, laboratories that are capable of performing internal quality control and standardization functions can save a considerable amount of money when evaluating cytokines. Sample Types and Preparation Cytokines are present in trace amounts in serum and plasma under normal circumstances. Fortunately, the current ELISA tests are sensitive to about 5 to 50 picograms of cytokine per ml, which is a useful range. Cytokine can be quantitated equally well in either serum or plasma. Levels of cytokines measured by ELISA are the same in both fluids. The test format (capture assay) eliminates most competitive substances present in these fluids. One important consideration is that the cytokines are not very stable and have short half-lives. Therefore, samples must be collected and tested fresh, or rapidly deep frozen for later evaluation. Freeze-thaw cycles tend to break down the cytokines and must be avoided. Most cytokine activity is stable for up to 8 hours in serum or plasma in a refrigerator. Isolated or purified cytokines (e.g., standards) are far less stable. 3 A capture assay is an immunoassay in which a microtiter plate is first coated with a "capture antibody" (usually polyclonal) to the antigen to be measured; the antigen-containing test solution is then applied, and antigen is captured by (bound to) the capture antibody. The captured antigen is then detected using a secondary antibody (often monoclonal) that is covalently bound to a substance that can be converted to a detectable species (for example, a biotinylated secondary antibody becomes detectable when reacted with enzyme-labelled streptavidin and appropriate substrate.)
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--> Clinical Use of Cytokines A large number of cytokine clinical trials (Phase I, Phase II and Phase III) are underway in the United States and throughout the world. The indications for such trials are wide-ranging, spanning the spectrum of diseases from cancer, to autoimmune conditions, inflammatory conditions, and infectious diseases due to bacteria, parasites and viruses (cf, Oppenheim et. al., 1993). Since cytokines have such broad regulatory influences on immunological responses, they could theoretically be applied in almost any case where host defenses need to be boosted or suppressed. Unfortunately, our understanding of the full scope of activities of individual cytokines, and of the interactions among the cytokines, is not sufficiently sophisticated to allow trials of mixtures of cytokines; the first trials involve careful observations following the administration of individual cytokines to patients who generally have failed all conventional modes of therapy. Several general observations may be made concerning these studies. First, as expected, cytokines administered parenterally in large amounts can result in numerous undesirable side effects, since the cytokine concentrations reached in body fluids during these trials far exceed levels normally observed. Major side effects have included fevers, loss of vascular integrity (capillary leakage) with accompanying hemodynamic problems, and flu-like syndromes. Since there are so many cytokines, and their properties and actions differ so greatly, it is difficult to generalize regarding side effects. The clinical approach to cytokine use is still basically an empirical one, and testing of new cytokines must be undertaken only with great caution. Even so, the use of some cytokines, such as the hematopoietic growth factors, is already routine for some uses (e.g., enhanced recovery of white blood cells following chemotherapy for cancer). Other approved indications will be added in the near future. Cytokines and Nutrition: Author's Observations and Recommendations The inclusion of an independent presentation on the topic of cytokine biology as related to nutritional status represents a major new direction of investigation in both immunology and nutrition. Although some information on cytokine responses to stresses such as exercise or infection is available (and, in fact, is liberally represented in data offered in this volume), there are as yet relatively few published studies (Gallagher and Daly, 1993; Grimble, 1995; Harbige, 1996). It is logical to assume that the cytokines, acting as the hormones of the immune system, would be affected generally by nutritional status in the same way that other physiological systems would be affected. For example, chronic nutritional deficiencies adversely affect the immune response, including reductions in the production and activity of cytokines. However, little
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--> information is available concerning whether cytokine deficits would occur gradually as nutritional adequacy becomes limiting, or whether there would be a threshold effect, where cytokines would suddenly cease to be produced at a certain level of nutritional deficit. Another way to look at the current data is to think about what can be done now, versus what can be done later, after more knowledge is acquired. Mechanisms are important and will need to be understood to get a full picture of the cytokine-nutrition interrelationship. However, considering the current status of knowledge in this area, more data probably need to be collected. "Phenomenology," often used as a pejorative in science, meaning that one is looking for descriptions and not for theory, may be the order of the day; theory will come later. It is not unlikely that the measurement of cytokines in an acute stress situation will offer some picture of the way the body is responding, especially if the stressors have been shown to impair general health. To date, the cytokines most closely examined have been those in the "inflammatory cytokine" group, since physical stress is well known to result in generalized and specific inflammation in the tissues. Exercise, for example, induces changes in cytokines such as IL-1 (see Nieman, Chapter 17 in this volume). As monitors of the body's attempts to achieve homeostasis, the cytokines may be good "leading indicators" of future health status. It would be straightforward to design a study to investigate this point, along the lines of existing studies on military trainees reported at this meeting. Malnutrition and problems related to subnormal caloric intake may be evident quite early by using cytokine levels as surrogate markers of health status. Since so little has been done to investigate this possibility, there is strong rationale for measuring many cytokines in a controlled study and then checking to see if a single cytokine or (more likely) a "profile" of cytokine activity is predictive of disease susceptibility and outcome. Another fruitful line of investigation would be to examine the in vitro ability of blood cells from various individuals (for example, military trainees) to respond to stimulators such as antigens (tetanus, influenza, etc.) or mitogens (phytohemagglutinin, concanavalin A) with appropriate cytokine responses. These data could be correlated with the ability of these individuals to cope with stresses such as malnutrition and strenuous physical activity. Perhaps cytokine responsivity can predict which individuals could withstand better the rigors of training, with less chance of infection and disease. This knowledge would be of value in choosing appropriate personnel for specific missions. No data currently exist that address this topic. In summary, the new area of cytokine biology, in which the so-called hormones of the immune system are monitored in order to assess disease resistance and general health, may be a fruitful area of study. The fact that the cytokines are short-lived, and that their production represents an accurate "snapshot" of current immune status, may prove valuable in assessing both the
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--> effects of experimental modification of diet and nutritional factors and interactions between diet and stresses such as exercise or fatigue in terms of impact on health and resistance. However, the field is so new that considerable groundwork needs to be done to assess the feasibility and applicability of cytokine measurement as an adjunct to other tools used to monitor nutrition and health. References Aggarwal, B.B., and R.K. Puri, eds. 1995. Human Cytokines: Their Role in Disease and Therapy. Cambridge, Mass.: Blackwell Scientific. Callard, R., and A.J.H. Gearing. 1994. The Cytokine Facts Book. London: Academic Press. Casciari, J.J., H. Sato, S.K. Durum, J. Fiege, and J.N. Weinstein. 1996. Reference databases of cytokine structure and function. Cancer Chemother. Biol. Response Modif. 16:315-346. Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds. 1994. Current Protocols in Immunology. New York: John Wiley & Sons. Gallagher, H.J., and J.M. Daly. 1993. Malnutrition, injury and the host immune response: Nutrient substitution. Curr. Opin. Gen. Surg. 93:92-104. Grimble, R.F. 1995. Interactions between nutrients and the immune system. Nutr. Health 10:191-200. Harbige, L.S. 1996. Nutrition and immunity with emphasis on infection and autoimmune disease. Nutr. Health 10:285-312. Kopp, W.C., and J.T. Holmlund. 1996. Cytokines and immunological monitoring. Cancer Chemother. Biol. Response Modif. 16:189-238. Kuby, J. 1994. Immunology. New York: W.H. Freeman and Company. Oppenheim, J.J., J.L. Rossio, and A.J.H. Gearing, eds. 1993. Clinical Applications of Cytokines: Role in Pathogenesis, Diagnosis and Therapy. New York: Oxford University Press. Whiteside, T.L. 1994. Cytokines and cytokine measurements in a clinical laboratory. Clin. Diagn. Lab. Immunol. 1:257-260. Discussion RONALD SHIPPEE: In our field studies, we typically draw blood quite a bit. Could you give us some guidance on handling those samples, how fast we have to get it out of the glass, suggested temperature, and surely regarding shipping samples. JEFFREY ROSSIO: Cytokines are small proteins, and they are relatively susceptible to proteolysis. They have relatively short half lives. In order to store cytokines themselves for shipment, if you are specifically looking for plasma cytokines, I think that the samples would have to be frozen fairly quickly. They are stable once frozen.
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--> RONALD SHIPPEE: In getting the cells separated from the plasma, are there guidelines? JEFFREY ROSSIO: In terms of what things to measure, there are two things we can look at. We can look at the capability of the cells to produce cytokines or we can look at a snapshot of what cytokines are already there. If you want to deal with the cells the way Tim Kramer is dealing with the cells, then I think it is fairly important to keep the cells in the milieu where they are happy (which is not too cold because cold inhibits the ability of the cells to recover their cytokine production) and to get them to the laboratory as quickly as possible. We have a lot of experience with shipping samples across the country, and if they are shipped across the country in the winter, even in those insulated Styrofoam Federal Express mailers, the cells lose almost all the biological activity when they get cold. ARTHUR ANDERSON: We actually have been able to measure two of the signal cytokines like IFN-γ or TNF. The IFN-γ and TNF activity was measured in serum that was shipped from Korea during the Korean War, and after all these years after the Korean War and being stored in a -70° walk-in freezer, we are still able to measure the INF-γ and TNF. JEFFREY ROSSIO: Had it been stored at -70° since its collection? ARTHUR ANDERSON: It had been stored at -70° but was shipped in wet ice. ROBERT NESHEIM: Any other questions or comments? STEVE GAFFIN: Is it known whether the cytokines pass through the gut wall? JEFFREY ROSSIO: I don't know. It is known that the gut makes, or the enterocyte makes, IL-4. STEVE GAFFIN: What about the permeability of the gut wall? JEFFREY ROSSIO: We don't think they are present in the lumen, but there is little data to address this point.
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--> ARTHUR ANDERSON: We fed cytokines to mice as an adjuvant for an oral vaccine study, and found that fed cytokines will increase immune response in a cytokine-appropriate way. So, you can have both. You can have enterocytes making cytokines and also cytokine absorption present. JEFFREY ROSSIO: One of the things in my manuscript that I didn't have time to talk about involves the intervention with cytokines; you have to remember that the cytokines are designed to be intercellular messengers. Most of the cytokines work at very short distances and for very short times. When cytokines are administered in large amounts in an adjuvant-type setting, the side effects of those cytokines are often very severe. We have a lot of experience with using cytokine therapy in cancer patients, and there are very major problems with administering nonphysiological or large amounts of cytokines, and that is something that will have to be overcome. The delivery problem is something that needs much more study. GERALD KEUSCH: I am thinking about the complexity of the issue of cytokines in the gut. In model systems using cultured human intestinal epithelial cells in regions where the membrane forms very nice tight junctions, they will make IL-8, but it is almost all at the basal side. IL-8 appears to have a very important physiological effect as a chemokine to attract neutrophils. The neutrophil response actually goes through the monolayer of epithelial cells and will facilitate the transfer of bacteria from the apical side to the luminal side. In shigellosis, the cytokine cascade including IL-8 and IL-1 in particular, is part of the pathogenesis of the disease. The inflammatory response of those cytokines is essential for creating the disease. So, we have heard about double-edged swords. Clearly at the level of the gut in intestinal disease, that is one of the critical infectious diseases in the military for fighting capacity. We have got to be very careful about understanding what happens when you put cytokines on the surface or just below the gut surface. JEFFREY ROSSIO: And depending on cytokine status, the balance of cytokines, especially the Th1-Th2 balance, may affect the outcome of the disease process depending on the pathogen. There is a lot of study in parasitology looking at leishmaniasis and other types of parasites where the Th1-Th2 cytokine balance, determines the outcome of the disease. DAVID NIEMAN: In regard to the use, let us say you are measuring total inflammatory cytokines in response to exertion. The lab that we have been
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--> working with is trying to get us to send them serum instead of plasma, but it is easier for us to use plasma. Do you have an opinion on which is better? JEFFREY ROSSIO: We have done a lot of measurements both ways. Plasma is usually easier to get and more available. We haven't had a lot of trouble in measuring cytokines in plasma. So, probably you have to talk to the laboratory and see if they have a problem with standardizing their assays one way, and they don't want to go to the trouble of standardizing them again or not. We haven't found interfering substances. Now, in some of our studies in cancer patients, we have gone to the extent of doing a double molecular filtration to remove everything with a molecular weight of over 50,000 and everything under a molecular weight of 15,000 or 10,000 with centrifugal filters to see if there were inhibitors around that were affecting our results. The conclusion of that study was that it was not worthwhile to go to the trouble of performing a double molecular filtration. DAVID NIEMAN: One follow-up question. Immediately after heavy exertion when we take a sample and separate the plasma and try to freeze it within 30 minutes, on doing that we find that IL-6 goes up about sixfold. Is there anything you can recommend like deliverance timing for freezing, or is 30 minutes okay? JEFFREY ROSSIO: I don't know. We usually just recommend as fast as possible. I think 30 minutes is probably okay. When cytokines are present in low concentrations, like any protein in low concentration, they have a tendency to adhere to glass, to adhere to polystyrene. So, we are usually trying to do it as quickly as possible and get it frozen as quickly as possible.
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