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Smoke Gets in Your Cells

Abbreviations: cigarette smoke extract, CSE

	Introduction

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In general, studies in this journal provide novel mechanistic insight into lung (patho)biology. These studies involve molecules (proteins, lipids, carbohydrates), cells, and/or whole organisms. Most individual studies focus on one of these areas, and a compilation of protein, cell, and animal-based manuscripts allows us to synthesize a more comprehensive view of the particular process. Studies that can attack a problem at multiple levels often have most scientific merit. For example, a cell culture study is strengthened by in vivo confirmation, and an in vivo finding is improved with mechanistic insight gained at the cell/protein level. It is this synergistic combination of in vivo and in vitro studies that is often most informative of underlying pathophysiologic mechanisms.

	Biological Agents

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To determine the effect of a particular biological agent on a cell, one frequently adds the agent to cells in culture and monitors specific cellular responses. One can add biologically relevant agents, such as lipopolysaccharide, or those that might activate a cell but have less pathophysiologic importance, like phorbol esters. When used, more complex mixtures, like cigarette smoke extract (CSE), have the advantage of containing all of the compounds inhaled by smokers. Yet the very complexity of CSE makes it difficult to identify the culprit mediating a specific effect. Arguably, it is of greater concern to determine the biological relevance of particular concentrations and durations of exposure of a given agent. To that end, one can perform a dose–response and time course and make some crude calculations to suggest that exposure mimics what might happen in vivo. However, even if one could assess exposure, such experiments cannot duplicate all of the components of the microenvironment—such as the mucus layer—that exist in living systems. Nonetheless, the goal of the cell system is to simplify the experiment and limit variables. That CSE differs from gaseous smoke makes it difficult to determine which dose and exposure time best recreate the effects of smoking on the lung. Yet, despite severe limitations, results from these studies allow researchers to determine the capacity of CSE to influence cellular functions while eliminating other variables. For example, we know that macrophage matrix metalloproteinases are induced in vivo after exposure to cigarette smoke, but one cannot determine if this is a direct effect of smoke on the macrophage or works indirectly by inducing another cellular product that works on the macrophage.

	Cell Culture

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What about the difficult but important and more general issue regarding choice of cells to use in one's study? In this issue of AJRCMB, the article by Kim and colleagues (beginning on page 483) (1) uses primary fetal fibroblasts. Fetal fibroblasts might have less relevance than adult fibroblasts here, yet at least these were primary cells. Most believe that there is greater merit in studies that use primary cells rather than cell lines. In fact, if one were to study airway epithelial biology, primary cells maintained in an air–liquid interface is the gold standard; yet one can certainly learn from simpler systems. Cell lines are transformed to proliferate indefinitely and, by virtue of their biology, often have subtle, if not profound, variations in signaling pathways. A cell line in late passage may have acquired mutations, shifting the genotype and subsequent phenotype. Moreover, a single cell line cannot be used to explore genetic diversity. Primary cells, on the other hand, may be perturbed during lengthy isolation, and their purity and yield may be limiting; however, the use of techniques such as cell sorting may reduce isolation time, and we now can do more with fewer cells. Primary cells may also be difficult to transfect, thus limiting the ability to directly manipulate the system. In fact, some may argue that primary cells are not necessarily any better than cell lines—just different. Perhaps a judicious combination of both primary cells and cell lines is best—primary cells to confirm the importance of findings, and cell lines to probe the mechanism in detail.

Attention must also be given to the species from which the cell line was derived. Are human cells better than mouse cells? While understanding human biology and treating human disease are our ultimate goals, cells from other organisms may have advantages over those from humans. Animal cell lines are often easier to obtain. And, in some instances, non-human cell lines provide better models than human cells. For example, mouse macrophage cell lines, such as P388D1, RAW, and J77 cells, are more differentiated than human cell lines, such as HL60 and U937. The latter require agents such as phorbol esters to differentiate them to macrophages, a process which complicates the issue by activating the cells (2). Trickier still, murine—but not human—macrophages produce large amounts of nitric oxide in response to stimulation (3). Therefore, knowledge of both the animal cell model and human biology are required to make translational inferences.

Perhaps the most widely used and criticized cell line is the A549, thought to be a model of alveolar type II epithelial cells. This line was initiated in 1972 through explant culture of lung carcinoma from a 58-yr-old Caucasian male (4). It shares all the advantages and disadvantages of a cell line discussed above. A Medline search (http://medline.cos.com/) of A549 cells lists over 2,500 publications. Use of these cells has led to many important discoveries, including several observations regarding cell signaling that have been very useful in the development of chemotherapy for lung cancer patients. However, it has also led to many dead-ends (both scientifically and career-wise). For example, A549 cells lack certain characteristics of differentiated cells, and do not form tight epithelial barriers with intact cell-to-cell junctions critical to fluid movement (5).

In summary, CSE has appropriately come under "fire" for the use of short-term, high concentrations of an undefined and homemade reagent to extrapolate to long-term, low concentrations inhaled by smokers. Nevertheless, like all imperfect tools, it has utility to uncover important biological pathways. Ideally, the discovery would be most convincing if it occurred in primary cells, the mechanism was explored in depth in a cell line (if necessary), and ultimately confirmed in vivo.

Given the complex issues regarding use of CSE, perhaps the simplest solution would be for humans to stop ingesting this dangerous substance. Remember, this is the only legal substance that, when used as directed, kills one-half of the users. One can only hope that future generations will make better choices. We also hope that this specific example helps readers/investigators with their choices of model systems.

	Footnotes

 
Conflict of Interest Statement: In the past three years, S. Shapiro has participated in Advisory Boards for Boehringer Ingelheim, GlaxoSmithKline, Millennium, Pfizer, Wyeth, and ICOS; he received compensation ranging from $1,500 to $7,500 for each. His laboratory has performed research in collaboration with Pfizer, Arriva, ONO, and Taisho, and received grants for these projects ranging from approximately $100,000 to $200,000; no personal income was received.

Received in original form August 27, 2004

	References

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1. Kim, H., X. Liu, T. Kobayashi, H. Conner, T. Kohyama, F.-Q. Wen, Q. Fang, S. Abe, P. Bitterman, and S. I. Rennard. 2004. Reversible cigarette smoke extract–induced DNA damage in human lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 31:483–490.[Abstract/Free Full Text]
2. Welgus, H. G., N. L. Connolly, and R. M. Senior. 1986. 12-o-Tetradecanoyl-phorbol-13-acetate–differentiated U937 cells express a macrophage-like profile of neutral proteinases: high levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G. J. Clin. Invest. 25:4750–4755.
3. MacMicking, J., Q. W. Xie, and C. Nathan. 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323–350.[CrossRef][Medline]
4. Giard, D. J., S. A. Aaronson, G. J. Todaro, P. Arnstein, J. H. Kersey, H. Dosik, and W. P. Parks. 1973. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 5:1417–1423.
5. Hermanns, M. I., R. E. Unger, K. Kehe, K. Peters, and C. J. Kirkpatrick. 2004. Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro. Lab. Invest. 84:736–752.[CrossRef][Medline]

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