Cytokines Play Growing Role As Research Reagents In Biological Labs

Molecular biologists and biochemists these days are focusing an increasing share of their investigations on the activities of a class of small peptide messenger molecules called cytokines. The following companies provide a broad array of cytokines and related products for use in biological research laboratories. For more information, please use the Reader Service Card inserted in this issue. Accurate Chemical & Scientific Corp. Westbury, N.Y. circle No. 049 on Reader Service Card AMAC/Immunotech Inc. Westbrook, Maine circle No. 050 on Reader Service Card Amersham Corp. Arlington Heights, Ill. circle No. 051 on Reader Service Card BACHEM Bioscience Inc. King of Prussia, Pa. circle No. 052 on Reader Service Card Becton Dickinson/ Falcon Labware Franklin Lakes, N.J. circle No. 053 on Reader Service Card BoehringerMannheim Biochemicals Inc. Indianapolis circle No. 054 on Reader Service Card Calbiochem Novabiochem Corp. San Diego circle No. 055 on Reader Service Card Chemicon International Inc. Temecula, Calif. circle No. 056 on Reader Service Card Endogen Inc. Cambridge, Mass. circle No. 057 on Reader Service Card Fitzgerald Industries International Concord, Mass. circle No. 058 on Reader Service Card Genzyme Diagnostics Cambridge, Mass. circle No. 059 on Reader Service Card Harlan Bioproducts for Science Indianapolis circle No. 060 on Reader Service Card Immunocorp Montreal circle No. 061 on Reader Service Card INAMCO Chemicals & Lab Equipment Flushing, N.Y. circle No. 062 on Reader Service Card INCSTAR Corp. Stillwater, Minn. circle No. 063 on Reader Service Card Intergen Co. Purchase, N.Y. circle No. 064 on Reader Service Card Life Technologies Inc. Gaithersburg, Md. circle No. 065 on Reader Service Card Mallinckrodt Chemical Inc. Chesterfield, Mo. circle No. 066 on Reader Service Card Oncogene Science Cambridge, Mass. circle No. 067 on Reader Service Card PerSeptive Diagnostics Cambridge, Mass. circle No. 068 on Reader Service Card PharMingen San Diego circle No. 069 on Reader Service Card Pierce Chemical Co. Rockford, Ill. circle No. 070 on Reader Service Card Promega Madison, Wis. circle No. 071 on Reader Service Card Quality Controlled Biochemicals Inc. Hopkinton, Mass. circle No. 072 on Reader Service Card R&D Systems Inc. Minneapolis circle No. 073 on Reader Service Card Research Diagnostics Inc. Flanders, N.J. circle No. 074 on Reader Service Card Sigma Immunochemicals St. Louis circle No. 075 on Reader Service Card StemCell Technologies Inc. Vancouver, B.C., Canada circle No. 076 on Reader Service Card Unisyn Technologies San Diego circle No. 077 on Reader Service Card VMRD INC. Pullman, Wash. circle No. 078 on Reader Service Card These incompletely understood substances, secreted by immune-system cells and transported in the blood, function chiefly as facilitators for a multitude of immune-system responses, such as inducing inflammation in targeted tissues or inhibiting the replication of viral invaders in the body. Receptors for these biochemical messengers are present on the surfaces of many types of cells throughout the body, and cytokines are important players in a wide array of cellular activities, both normal and pathologic. For example, genetically modified mice--so-called knockout mice--that lack genes for cytokines or cytokine receptors generally die either before or shortly after birth, implying, among other things, that cytokines play critical roles during the development process. Other areas currently under study include the biology of endothelial cells, vasculogenesis, angiogenesis, and atherosclerosis. In the last decade, an avalanche of such research findings about cytokines has led to a booming demand for these molecules as reagents to facilitate study of their unique properties. The list of commercially available purified cytokines is growing exponentially, with suppliers of laboratory biochemicals working closely with researchers in producing cytokines that may have uses in investigations of cancer, arthritis, HIV, and other diseases. In cell and developmental biology, an especially active area of investigation concerns the stimulatory effects of cytokine growth factors on cells in culture. "Ninety to 95 percent of the calls we receive are from researchers who are trying to stimulate the growth of a particular type of cell, such as a nerve cell or a particular hybridoma clone, with a growth factor," says Willis Eubanks, a technical specialist in cell biology for Life Technologies Inc. in Gaithersburg, Md. Cytokines help increase the production of antibodies from hybridoma cells, for example. Typically, the hybridoma cells are kept in a hollow-fiber growth chamber, separate from their secretions, to increase yield. "By stimulating the growth of the hybridoma clone there is a higher yield of antibody," Eubanks says. Life Technologies markets cytokine growth factors in association with tissue-culture media and reagents through its parent company, Gibco BRL in Grand Island, N.Y. Gibco also sells an array of cell-culture media, serum, and growth factors. Cytokines, with their wide range of biological activity, are among the most intriguing and actively studied members of the multicomponent system responsible for the body's strategic defense against disease. Other members of this complex network of cells and cellular biochemicals include white blood cells--some, such as macrophages, scavenge rogue particles and clear them from the body, while others secrete antibodies to neutralize toxic substances. Cytokines secreted by immune-system cells scurry between these cells bearing messages to step up the attack or slow it down. Although the list of identified cytokines is long, researchers have only begun to appreciate how complex this interactive network of regulatory substances really is. "Cytokines perform a myriad of different functions in the body," says Jordan Fishman, director of scientific operations and cofounder of Quality Controlled Biochemicals Inc. (QCB) in Hopkinton, Mass., a biochemical reagents supply company. "Generally, cytokines act as intercellular messengers, particularly in response to an infection. They affect cells in many of the major organ systems as well as the immune system, where they cause the proliferation and differentiation of immune cells like T cells and the various T-cell subsets, B cells, and macrophages." QCB is another of the growing number of companies that sell cytokine products such as human cytokines, mouse cytokines, and cytokine assay reagents. But QCB, along with companies such as R&D Systems of Minneapolis and Endogen Inc. in Cambridge, Mass., also offer more specialized items. Endogen, for example, markets three principal product lines, consisting of reagents and immunoassay test kits to human and mouse cytokines, and for cell-adhesion studies. Cytokines were initially recognized in the mid-1960s as potent mediators of cell division and differentiation. Lymphocytes in culture could be stimulated to produce these biologically active substances when they were treated with specific antigens or with nonspecific mitogens such as poke weed, which induces many kinds of cells to proliferate. Early investigators characterized these mysterious substances by the functional response they gave. The scientific literature was peppered with reported discoveries of a large number of cell factors with stimulatory or inhibitory activities, bearing names like lymphocyte-activating factor (LAF), T-cell growth factor (TCGF),B-cell-activating factor (BAF), and mitogenic factor (MF). Once the individual factors were purified and cloned, researchers found that many of the reported activities could actually be traced to a much smaller number of substances, which were all small-molecular-weight proteins apparently secreted by various immune-system cells. As a group, these secreted biochemicalswere eventually dubbed "cytokines," to denote their interactive role in cellularcommunication. "In a general sense, growth factors, interferons, and interleukins could all be categorized as cytokines," says Eubanks of Life Technologies. "The confusion in the nomenclature stems from the field growing so rapidly. If you look at the history of an individual cytokine, you might see it referred to as a specific growth factor or stimulating factor before it became known as an interleukin." Cytokines act by binding to a receptor molecule on the surface of a cell, causing an intracellular message to be transduced across the cell membrane. Although the precise mechanisms involved are not completely understood, the transduced signal appears to be able to activate the genes responsible for cell division, differentiation, or other cell processes. In many cases, the cytokine message stimulates target cells to produce more and different kinds of cytokines. Sometimes, a cytokine can stimulate certain cells to divide or differentiate, while at the same time deactivate other cells involved in the same immune response. "Cytokines complement each other," says Eubanks. "One cytokine might stimulate one type of cell where another might suppress it." For example, says Albert Millis, a cell biologist at the State University of NewYork, Albany, "TGF-B has been shown to induce the expression of connective tissue-like molecules, while at the same time suppressing or inhibiting the expression of certain metalloproteinases that degrade this type of tissue." Millis uses cytokines like TGF-B in his studies on fibroblasts and smooth muscle cells and notes that, at least in vitro, these effects seem to be concentration-dependent."At one concentration TGF-B appears to be an inhibitor, while at other concentrations this same cytokine stimulates growth." The concentration dependency of cytokines may have something to do with the extraordinary potency of these small proteins. "Cytokines act in the picomolar to femtomolar range," says Fishman. "Even at very low concentrations, their effect is profound." Because cytokine receptors are expressed on the surfaces of such a large number of diverse cells, the response elicited by these chemical messengers is often swift and far reaching. And they seldom act alone. In an immune response to a foreign particle, for example, a single lymphocyte might secrete several different cytokines into the extracellular mileu. How target cells respond to the secreted mix of cytokines varies. Some cells proliferate, others might produce antibodies, while still other cells secrete additional cytokines that call phagocytic scavengers into the fray. Ultimately, a complex network of interacting immune cells is generated that effectively disarms the offending particle. Once the battle is over, additional cytokines signal the immune system to turn down the response and get back to daily business. Lymphocytes and macrophages aren't the only cells under the influence of the cytokine network. Researchers L. Maximilian Buja and Roger J. Bick at the University of Texas Health Science Center in Houston have added cytokines to cultures of cardiac cells, or myocytes, and observed how the cells and substances interact during periods when the myocytes are stressed, such as in the case of reduced blood flow to the heart, or ischemia. In their studies, cytokines IL-1a and IL-1o, along with TNF, were found to modulate the activity of the myocytes, perhaps playing a key role in maintaining cardiac function during periods of acute stress. Many researchers believe that there is logic in this natural strategy. "Even things like liver regeneration have been shown to be influenced by cytokines," says QCB's Fishman. RTo a point, the proliferation or differentiation of a cell is going to be similar regardless of the cell type. If you can modify the activity of a blood cell [with cytokines], you should be able to do the same thing with a parenchymal cell or a hepatocyte, and studies are proving that it works." Several cytokines are known to be involved in regulating the body's inflammatory response. In acute inflammation, cytokines IL-1, IL-6, and TNF-a are released from tissue macrophages and, acting together, cause the body to respond with a plethora of biological activities, including the release of more cytokines and other biochemicals with greater systemic influence. The net effect might be fever, increased vascular permeability to the inflamed area, production of new platelets, and the synthesis of antibodies, along with other responses, all aimed at killing off an invader or healing the inflamed tissue. In the case of a chronic infection, as is sometimes seen with some infectious agents and in several autoimmune disorders, this form of massive response can go too far, causing cytokine- mediated damage to the surrounding tissue. The destruction of joint tissues in diseases like arthritis begins this way, for example. Because cytokines play such an important role in the overall functioning of the immune system, many researchers are looking at ways of using cytokines or cytokine inhibitors as human therapeutic agents. In the body, cytokines have been praised as potent viral and cancer-fighting agents, and conversely accused of promoting arthritis and severe allergic attacks. Thus cytokine research is multipronged--in some studies the goal is to boost cytokine production, while in others it is to turn it down or off. Research opinion is mixed over using cytokines as human therapeutics. For a time in the 1980s, many investigators were hopeful that certain cytokines could be used to treat some cancers. When tumors were found to draw the attention of the immune system, particularly natural killer cells, researchers turned to the potent properties of cytokines to enhance the body's inherent anticancer response. Clinical trials of diverse interleukins, interferons, and TNF have shown varying degrees of success. In in some people, tumors regress; but in other patients, regression is partial or undetectable. Moreover, the cytokine approach to tumor treatment has been fraught with technical problems, including the short lifespan of the soluble substances along with the variety of adverse side effects brought on by cytokine treatments. Because of the variations in the results of these types of studies, the focus of cytokine research along these lines has switched to the test tube. Using cytokines to activate cells taken from a cancer patient and grown in culture has shown promise, especially for inoperable tumors. When the cytokine-treated cells are infused back into the cancer patient, the activated cells target the tumor for destruction by the immune system. Another approach is gene therapy. Cells removed directly from a tumor can be supercharged with extra cytokine genes with the hope that the engineered tumor cells will produce cytokines to step up the defense effort by attracting the patientUs own immune cells. "The immune system is very powerful," says Fishman, "and cytokines are very potent modulators of the immune response." A cytokine-focused immune attack could selectively kill off the cancerous cells, leaving the healthy tissue intact. Viral infections are known to induce specific patterns of cytokine production, particularly from T-cells. Some cytokines produced in response to HIV infection have been implicated at many levels in the pathology of AIDS. On the other hand, for viruses like HIV, which hide inside of cells, a T-cell-mediated response may be the only way for the immune system to bring an infection under control. It may be possible to use cytokines to selectively stimulate the subsets of lymphocytes that fight viral diseases like AIDS. "The immune system doesn't always do the right job," concludes QCB's Fishman. "But perhaps with the right combination of cytokines we could achieve `super-human' results." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Cytokines Play Growing Role As Research Reagents In Biological Labs

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