Isolation and immunophenotyping of mononuclear cells from human lung tissue.
OBJECTIVE
To quantitatively isolate and immunologically phenotype mononuclear cells contained in human lung tissue.


METHODS
Normal appearing lung tissue as far distal to the resected lesion as possible was obtained from lung cancer patients. Lung tissue was thoroughly washed and cut into small pieces and digested with collagenase. Peripheral blood mononuclear cells (PBMNC) were prepared from controls using Ficoll gradient. Isolated cells and PBMNC were analyzed by flow cytometry. We immunohistochemically stained snap-frozen lung tissue with anti-CD3, CD4, CD8, CD20, and CD161 antibodies.


METHODS
Nineteen patients with lung cancer who underwent lobectomy were enrolled. Twelve healthy volunteers also participated as controls for flow cytometric analysis of PBMNC.


RESULTS
In forward scatter vs side scatter, 92.1+/-7.8% of isolated cells in the lymphoid population expressed leukocyte common antigen, CD45. The frequency of CD45-positive cells in the lymphoid population from lung tissue was as high as that from PBMNC (p=0.118). CD45-positive cells were successfully further extended by anti-CD3, CD4, CD8, CD19, and CD161 antibodies. Monocyte-macrophages bearing CD68 were also detected. CD68-positive alveolar macrophages disappeared from alveolar spaces after thorough washing by immunohistochemical staining. Mononuclear cells in the interstitium were positively stained by anti-CD3, CD4, CD8, CD20, and CD161 monoclonal antibodies.


CONCLUSIONS
We could isolate interstitial cells and analyze cell surface markers via flow cytometry from fresh lung specimens by collagenase digestion without further purification. Immunohistochemistry confirmed the presence of the cells detected by flow cytometry in the lung interstitium.
Shimizu
Takashi
T
The Division of Respiratory Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
Fujimori
Fumio
F
Shimaoka
Yuichi
Y
Narita
Jun-ichi
J
Takada
Toshinori
T
Tajima
Shunji
S
Moriyama
Hiroshi
H
Terada
Masaki
M
Suzuki
Eiichi
E
Gejyo
Fumitake
F
D000368:Aged; D000369:Aged, 80 and over; D018951:Antigens, CD20 / Q000276:immunology; D001707:Biopsy, Needle; D015704:CD4 Antigens / Q000276:immunology; D016827:CD8 Antigens / Q000276:immunology; D002289:Carcinoma, Non-Small-Cell Lung / Q000276:immunology / Q000473:pathology*; D016022:Case-Control Studies; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000473:pathology; D008175:Lung Neoplasms / Q000276:immunology / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D011336:Probability; D012016:Reference Values; D012680:Sensitivity and Specificity; D046509:Tissue Culture Techniques
D018951:Antigens, CD20; D015704:CD4 Antigens; D016827:CD8 Antigens; D017493:Leukocyte Common Antigens