TABLE 1.

IFN-γ IS PRODUCED BY NEUTROPHILS IN 24-HOUR S. pneumoniae PNEUMONIA

				Percentage of Cells Producing IFN-γ
Site of Cells	Lineage-specific Marker	Lineage	Duration of Pneumonia (h)	PBS (Control)	S. pneumoniae
Lung digests	CD45	All leukocytes	6	0.09 ± 0.02, n = 3	0.10 ± 0.05, n = 3
			24	0.22 ± 0.07, n = 8	14.2 ± 0.95,* n = 14
	CD3	T cells	24	0.23 ± 0.06, n = 3	0.47 ± 0.10,† n = 12
	CD45+, Ly-6G−	Nonneutrophilic leukocytes	24	ND	0.31 ± 0.04,† n = 3
	CD45+, Ly-6G+	Neutrophils	24	ND	21.98 ± 3.43, n = 6
BAL fluid	F4/80	Macrophages	24	ND	0.08 ± 0.11, n = 3
	Ly-6G	Neutrophils	24	ND	16.4 ± 1.5,‡ n = 3
Circulating blood	Ly-6G	Neutrophils	6	0.02 ± 0.04, n = 3	0.04 ± 0.02, n = 3
			24	0.20 ± 0.20, n = 3	0.32 ± 0.16, n = 3

Definition of abbreviations: BAL = bronchoalveolar lavage; ND = not determined; PBS = phosphate-buffered saline; S. pneumoniae = Streptococcus pneumoniae.

Flow cytometry was performed to detect intracellular IFN-γ levels in all leukocytes (CD45⁺ cells), T cells (CD3⁺ cells), nonneutrophils (CD45⁺Ly-6G⁻ cells), and neutrophils (CD45⁺Ly-6G⁺) in the lungs of S. pneumoniae–infected or PBS-treated mice 6 or 24 hours after instillation. Isotype-matched control antibody was used to assess the level of background staining and to set gates. The percentages of cells in each quadrant and the means of the percentage in the double-positive quadrant were calculated for the number of animals indicated. IFN-γ expression was similarly measured in F4/80- and Ly-6G–positive cells from the BAL fluid and circulating blood (n = 6 in each group). Neutrophils in the lung digests or the BAL fluid from pneumonic lungs at 24 hours expressed IFN-γ. None of the other leukocyte subtypes were found to produce IFN-γ using this approach. Data represent means ± SEM, and the number of mice (n) is provided for each study. The results are from three to six separate experiments.

^*P < 0.001 versus the PBS group.

^†P < 0.01 versus the CD45 group.

^‡P < 0.05 versus the F4/80 group.