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Communications to the Editor |

Choosing the Appropriate ControlChoosing the Appropriate Control FREE TO VIEW

Peter E. Morris, MD, FCCP; Elizabeth A. Fitzpatrick, PhD
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Affiliations: Assistant Professor Division of Pulmonary/Critical Care Medicine Department of Microbiology and Immunology University of Kentucky Lexington, KY ,  Department of Intensive Care Department of Pathology Kyoto Prefectural University of Medicine Kyoto, Japan



Chest. 1999;115(4):1214-1215. doi:10.1378/chest.115.4.1214-a
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We recently read the article by Kobayashi and colleagues,“ Expression of Inducible Nitric Oxide Synthase and Inflammatory Cytokines in Alveolar Macrophages of ARDS Following Sepsis” (June 1998).1 The article concerns the fluorometric determination of intracellular alveolar macrophage inducible nitric oxide synthase (iNOS), interleukin-1β, interleukin-6, and interleukin-8.

Dr. Kobayashi and colleagues1 conclude from their data that there is strong expression of iNOS in alveolar macrophages from ARDS patients. It is our opinion that in their report, the increase in immunostaining of alveolar macrophages in the ARDS group might represent nonspecific staining of their anti-iNOS antibody. It is true that the application of the anti-iNOS antibody was associated with an increase in staining in the ARDS group as compared to the control group. This result could be true of any antibody. The investigators should demonstrate that the increase holds when comparing the specific anti-iNOS antibody to a nonspecific antibody within the ARDS group. We respectfully suggest that further experiments be performed with a nonspecific antibody of the same subclass as the anti-iNOS antibody included. With these control experiments, a conclusion could be made that references the nature of the specific anti-iNOS antibody’s pattern of staining in ARDS alveolar macrophages as compared to the pattern of the nonspecific-isotype antibody with a similar fluorochrome.

Correspondence to: Peter E. Morris, MD, FCCP, Division of Pulmonary/Critical Care Medicine, 800 Rose Street, MN 614, Lexington, KY 40536–0084

Kobayashi, A, Hashimoto, S, Kooguchi, K, et al (1998) Expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis.Chest113,1632-1639. [CrossRef]
 
To the Editor:

We thank Drs. Morris and Fitzpatrick for their comments about our manuscript, “Expression of Inducible Nitric Oxide Synthase and Inflammatory Cytokines in Alveolar Macrophages of ARDS Following Sepsis” (June 1998).1-1

They pointed out the importance of control experiments, especially focusing on the nonspecific fluorescence of activated macrophages. We, of course, did control experiments routinely using both nonimmunized sera and Tris-buffered saline solution. Unfortunately, we have no data from experiments using absorbed antibodies. As a result, we did not observe significant nonspecific fluorescence within macrophages, except for coarse granular fluorescence within “dust cells” showing very wide fluorescent spectrum. This kind of fluorescence was easily distinguished from fluorescein isothiocyanate (FITC) fluorescence. The procedure was always conducted under supervision of an experienced pathologist (Y.U.).

In our Table 2, macrophages of ARDS patients with sepsis did not always show positive staining. This finding meant another kind of control experiment that supports the fact that our immunofluorescent staining was not nonspecific. Of course, we do not know whether or not commercially available primary antibodies used were really specific.

To avoid the contamination of nonspecific fluorescence in the measured value, we did not display fluorometric values in this article, but we showed percentages of positive-staining cells under fluorescent microscopy. So, our data are not quantitative, but qualitative.

Drs. Morris and Fitzpatrick also pointed out that the activated alveolar cells in the FITC wavelength probably fluoresce in greater intensity than control cells. We have no experience in measuring autofluorescence of neutrophils and macrophages by flow cytometry using FITC filter set. As the fluorescent spectra of autofluorescence mainly derived from nicotinamide adenine dinucleotide phosphate are excitation = 350 nm and emission = 460 nm, we could not understand what they meant. In our experience, the static cytometer is not sensitive enough to measure autofluorescence of neutrophils and macrophages even when using an appropriate selection of filter unit. For such an experiment, we have another set of fluorescent microscopy equipped with cooled charge coupled device camera and light pass for UV light. In addition, the shapes of the cells are quite different in flow cytometric sample and in static cytofluorometric sample, ie, round in flow cytometry and very flat in static cytofluorometry. The difference in the cellular thickness may cause the difference of observed fluorescence pattern. So, cytoplasmic diffuse fluorescence in cytocentrifuged cells will be very weak compared with flow cytometric specimen. These may be the reasons why we could not observe any autofluorescence brighter than the background one. If we use a flow cytometer in the future, we will remember their kind advice and will use longer wave-length dye instead of FITC.

We also have unpublished data (1998) that these activated alveolar macrophages in ARDS patients with sepsis show significant expression of inducible nitric oxide synthase messenger RNA. This finding also supports the result of this article.

Unfortunately, our clinical findings did not show any significant role of inducible nitric oxide synthase in the pathogenesis of ARDS. We hope further investigation by earnest researchers like Morris and Fitzpatrick will elucidate the significance of this finding.

Correspondence to: Atsuko Kobayashi, MD, PhD, Kyoto Prefectural University of Medicine, Kyoto, 602-0841, Japan; e-mail: satoru@koto.kpu-m.ac.jp

References
Kobayashi, A, Hashimoto, S, Kooguchi, K, et al Expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis.Chest1998;113,1632-1639. [CrossRef]
 

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References

Kobayashi, A, Hashimoto, S, Kooguchi, K, et al (1998) Expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis.Chest113,1632-1639. [CrossRef]
 
Kobayashi, A, Hashimoto, S, Kooguchi, K, et al Expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis.Chest1998;113,1632-1639. [CrossRef]
 
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