Characterizing the Effect of Pentamidine Isethionate on the Immune System Using Mouse Splenocytes as an Experimental Model

Figures & data

TABLE 1 Baseline relative percentages of positive cells for and mean fluorescence intensity (MFI) of antigens evaluated in murine splenocytes

Marker	Strain	% Cells	MFI
CD4^+	ICR	24.26 ± 1.36	109.03 ± 14.21
	BALB/c	21.16 ± 3.35	95.20 ± 3.67
CD8^+	ICR	29.69 ± 3.92	45.68 ± 12.35
	BALB/c	20.86 ± 1.41	32.70 ± 1.55
CD14^+	ICR	2.95 ± 0.48	35.24 ± 2.43
	BALB/c	4.07 ± 0.56	44.76 ± 2.98
CD19^+	ICR	13.38 ± 3.76	2271.49 ± 334.59
	BALB/c	23.37 ± 2.21	2109.16 ± 578.52

Splenocytes obtained by spleen desegregation were immediately labelled with monoclonal antibodies specific for CD4, CD8, CD14, and CD19 co-receptors and the marker expression then analyzed by flow cytometry. The results correspond to data obtained from CellQuest software and represent the mean (±SD) percentages obtained from the quadrants in each density plot that contained the respective positive samples for the evaluated marker. All results were generated from analysis of duplicate samples from each mouse; total mice analyzed (N) were 9 and 11 for the ICR and BALB/c strains, respectively. *Significant differences between the two mice strains (p < 0.05).

FIG. 1 Functional assay of blastogenesis with ICR and BALB/c spleen cells treated ex vivo with pentamidine. Splenocytes (2 × 10⁵) were placed into wells of a 96-well plate containing various concentrations of the drug. After 72 hr of incubation, the medium was removed and the cells were pulsed with 1 μ Ci [³H]-thymidine/well in fresh drug-free medium. Cultures were harvested 16 hr later and incorporated radiation was quantified. Data are reported in terms of counts per minute. All results were generated from analysis of triplicate samples for each mouse; total mice analyzed (N) were 9 and 11 for the ICR and BALB/c strains, respectively.

TABLE 2 CD4 cell mortality

Strain	Treatment	24 hr	48 hr	72 hr
ICR	Negative control	12.26 ± 3.38	31.85 ± 22.92	40.35 ± 4.81
	Pentamidine 50 μ g/ml	13.94 ± 0.52	21.01 ± 1.53	33.96 ± 0.01
	Pentamidine 200 μ g/ml	8.56 ± 5.06	16.18 ± 6.42	42.16 ± 2.90
BALB/c	Negative control	16.30 ± 6.58	23.68 ± 0.23	24.78 ± 4.40
	Pentamidine 50 μ g/ml	11.03 ± 2.31	17.36 ± 5.85	26.95 ± 21.55
	Pentamidine 200 μ g/ml	10.40 ± 0.31	26.93 ± 19.47	30.59 ± 0.22

Splenocytes were isolated, cultured with different drug concentrations, and labelled every 24 hr with monoclonal antibody specific for CD4 co-receptor. 7AAD was used as a viability label. All markers were then monitored by flow cytometry. The results correspond to data obtained from CellQuest software and represent the mean (± SD) percentage of non-viable cells among all the CD4⁺ cells (pre-selected in this gate) present from all performed replicates from all mice of a given strain. All results were generated from analysis of duplicate samples for each mouse; total mice analyzed (N) were 9 and 11 for the ICR and BALB/c strains, respectively.

TABLE 3 CD8 cell mortality

Strain	Treatment	24 hr	48 hr	72 hr
ICR	Negative control	10.50 ± 4.69	14.38 ± 2.90	22.20 ± 0.39
	Pentamidine 50 μ g/ml	5.68 ± 0.86	15.55 ± 4.63	19.71 ± 1.52
	Pentamidine 200 μ g/ml	7.64 ± 0.19	12.72 ± 7.54	25.58 ± 2.80
BALB/c	Negative control	5.58 ± 0.07	10.95 ± 0.13	20.96 ± 0.30
	Pentamidine 50 μ g/ml	10.71 ± 5.31	22.32 ± 10.76	20.48 ± 6.48
	Pentamidine 200 μ g/ml	11.16 + 0.18**	18.56 ± 6.02	22.50 ± 5.15

Splenocytes were isolated, cultured with different drug concentrations, and labelled every 24 hr with monoclonal antibody specific for CD8 co-receptor. 7AAD was used as a viability label. All markers were then monitored by flow cytometry. The results correspond to data obtained from CellQuest software and represent the mean (± SD) percentage of non-viable cells among all the CD8⁺ cells (pre-selected in this gate) present from all performed replicates from all mice of a given strain. All results were generated from analysis of duplicate samples for each mouse; total mice analyzed (N) were 9 and 11 for the ICR and BALB/c strains, respectively. ** p < 0.01; pentamidine-treated splenocytes vs. respective negative control (splenocytes in pentamidine-free medium). In addition, the cells that received 200 μ g/ml pentamidine show significant (p < 0.05) interstrain differences in CD8 mortality at 24 hr.

TABLE 4 CD19 cell mortality

Strain	Treatment	24 hr	48 hr	72 hr
ICR	Negative control	13.65 ± 0.18	19.76 ± 5.20	44.04 ± 0.00
	Pentamidine 50 μ g/ml	34.62 ± 31.72	23.91 ± 4.82	38.79 ± 5.86
	Pentamidine 200 μ g/ml	13.44 ± 1.74	21.37 ± 9.70	44.39 ± 0.91
BALB/c	Negative control	7.66 ± 0.21	17.64 ± 1.46	25.69 ± 0.97
	Pentamidine 50 μ g/ml	12.14 + 3.13	29.87 ± 12.62	27.09 ± 10.49
	Pentamidine 200 μ g/ml	12.69 ± 0.21**	26.49 ± 9.50	28.60 ± 5.85

Splenocytes were isolated, cultured with different drug concentrations, and labelled every 24 hr with monoclonal antibody specific for CD19 co-receptor. 7AAD was used as a viability label. All markers were then monitored by flow cytometry. The results correspond to data obtained from CellQuest software and represent the mean (± SD) percentage of non-viable cells among all the CD19⁺ cells (pre-selected in this gate) present from all performed replicates from all the mice of a given strain. All results were generated from analysis of duplicate samples for each mouse; total mice analyzed (N) were 9 and 11 for the ICR and BALB/c strains, respectively. ** p < 0.01; pentamidine-treated splenocytes vs. respective negative control (splenocytes in pentamidine-free medium).