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
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
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
Correspondence to: Peter E. Morris, MD, FCCP, Division of
Pulmonary/Critical Care Medicine, 800 Rose Street, MN 614, Lexington,
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
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:
Become a CHEST member and receive a FREE subscription as a benefit of membership.
Individuals can purchase this article on ScienceDirect.
Individuals can purchase a subscription to the journal.
Individuals can purchase a subscription to the journal or buy individual articles.
Learn more about membership or Purchase a Full Subscription.
Institutional access is now available through ScienceDirect and can be purchased at myelsevier.com.
Some tools below are only available to our subscribers or users with an online account.
Download citation file:
Web of Science® Times Cited:
Customize your page view by dragging & repositioning the boxes below.
Enter your username and email address. We'll send you a reminder to the email address on record.
Athens and Shibboleth are access management services that provide single sign-on to protected resources. They replace the multiple user names and passwords necessary to access subscription-based content with a single user name and password that can be entered once per session. It operates independently of a user's location or IP address. If your institution uses Athens or Shibboleth authentication, please contact your site administrator to receive your user name and password.