PMC

DCs inhibit TRALI-associated physiological responses. (A-D) Rectal temperatures (A), MIP-2 levels (B), pulmonary PMN percentages (C), and lung MPO enzyme activity (D) of either naive WT C57BL/6 mice or CD11c-DTR mice that were injected (+) or not (−) with the indicated reagents or antibodies. All mice were analyzed 90 minutes after the second injection. For the statistical analyses, only significant comparisons of interest are shown. The comparisons shown in panels A,C-D were analyzed by a 1-way ANOVA with a Tukey’s post hoc test. Each dot represents 1 mouse, and error bars represent SD. *P < .05, ****P < .0001.

Tregs inhibit TRALI-associated physiological responses. (A) Rectal temperatures of naive BALB/c mice or mice first treated (+) or not (−) with the indicated antibodies for Treg depletion and then injected with the TRALI-inducing antibody 34-1-2s or indicated control antibodies. (B) Pulmonary PMN percentages in the BALB/c mice treated in panel A. (C) MPO activity in the lung tissue of Treg-depleted BALB/c mice treated in panel A. (D) Plasma MIP-2 levels of BALB/c mice treated in panel A. All mice were analyzed 90 minutes after the second injections. For the statistical analyses, only significant comparisons of interest are shown. Panels A-D were analyzed with a 1-way ANOVA with Tukey’s post hoc test. Each dot represents 1 mouse, and error bars represent SD. *P < .05, **P < .01, ****P < .0001.

PMNs and ROS are required for antibody-mediated TRALI induction. (A) Lung W/D weight ratios of WT C57BL/6 mice or gp91phox KO mice which first underwent in vivo depletions of CD4⁺ T cells and/or PMN by the indicated antibody injections (+) or not (−) and were then injected with the TRALI-inducing antibody cocktail 34-1-2s + AF6-88.5.5.3 or indicated control antibodies. (B-E) Rectal temperatures (B), plasma MIP-2 levels (C), pulmonary PMN percentages (D), and lung MPO enzyme activity (E) in CD4⁺ T cell-depleted WT C57BL/6 mice with or without PMN depletion or gp91phox KO mice injected (+) or not (−) with the indicated antibodies. All mice were analyzed 90 minutes after the second injections. For the statistical analyses, only significant comparisons of interest are shown. All panels were analyzed by a 1-way ANOVA with Tukey’s post hoc test. Each dot represents 1 mouse, and error bars represent SD. *P < .05, **P < .01, ****P < .0001.

Tregs and DCs suppress TRALI by stimulating secretion of IL-10, a novel therapeutic agent. (A) Plasma IL-10 levels in naive BALB/c mice or mice first treated (+) or not (−) with the indicated antibodies for Treg depletion and then injected with the TRALI-inducing antibody 34-1-2s or indicated control antibodies. (B) Plasma IL-10 levels of naive C57BL/6 mice or CD11c-DTR mice injected (+) or not (−) with the indicated reagents or antibodies. (C) CD4⁺CD25⁺FoxP3⁺ Treg levels in spleen and thymus and CD11c⁺ DCs in spleen of the same C57BL/6 mouse injected with isotype mouse IgG2a or 34-1-2s + AF6-88.5.5.3 (TRALI resistant). (D) Plasma IL-10 levels in WT C57BL/6 mice or IL-10 KO mice treated 34-1-2s + AF6-88.5.5.3. (E-I) Lung W/D weight ratios (E), body temperature (F), MIP-2 levels (G), pulmonary PMN percentages (H), and lung MPO enzyme activity (I) of WT C57BL/6 mice treated with 34-1-2s + AF6-88.5.5.3 or IL-10 KO mice treated with either isotype mouse immunoglobulin G2a (IgG2a) or 34-1-2s + AF6-88.5.5.3. (I) Lung W/D weight ratios of CD4⁺ T-cell–depleted C57BL/6 mice (J), Treg-depleted BALB/c mice (K), and DC-depleted CD11c-DTR mice (L) infused with 34-1-2s + AF6-88.5.5.3 and treated prophylactically with (+) or not (−) with murine IL-10 administration (45 µg/kg IV). (M) Lung W/D weight ratios of CD4⁺ T cell depleted C57BL/6 mice, infused with 34-1-2s + AF6-88.5.5.3, and treated therapeutically 15 minutes later with (+) or without (−) murine IL-10 administration (45 µg/kg IV) after onset of TRALI (at least 2-degree drop in rectal temperature 10 minutes after TRALI-antibody injection). All mice were analyzed 90 minutes the second injection. For the statistical analysis, only significant comparisons of interest are shown. The comparisons shown in panels A-B,E-I were analyzed a with 1-way ANOVA with Tukey’s post hoc test; panels D,J,L-M were analyzed with 1-tailed unpaired t test; and panel K was analyzed with a 1-tailed Mann-Whitney test. Each dot represents 1 mouse, and error bars represent SD. *P < .05, **P < .01, ***P < .001, ****P < .0001.

DCs convey strong protection against antibody-mediated TRALI. (A) Representative flow cytometric dot plot analysis of splenocytes showing in vivo depletion of CD11c^high+ DCs (from 0.81% to 0.10% of total spleen cells) compared with isotype staining. (B) Representative macroscopic images of lungs from an in vivo DC-depleted mouse 90 minutes after injection of either a mouse immunoglobulin G2a (IgG2a) isotype control antibody (left) or 34-1-2s + AF6-88.8.8.3 (right). (C) Lung W/D weight ratios of WT C57BL/6 mice or CD11c-DTR mice that first underwent in vivo depletions of DC by the indicated injections (+) or not (−) of DT and were then injected with the TRALI-inducing antibody cocktail 34-1-2s + AF6-88.5.5.3 or indicated control antibodies. (D) Lung histology from naive WT C57BL/6 mice (subpanel A1-A2) and CD11c-DTR mice (subpanels B-E) receiving the indicated antibody injections. Subpanels A1-E1 and A2-E2 represent lung tissue images taken at original magnification ×20 and ×40, respectively. Representative images of each indicated group are shown. A zoom of indicated square in subpanel E2 (lung section from mice depleted of DCs and injected with 34-1-2s + AF6-88.5.5.3) is depicted and shows alveolar PMN infiltration. Scale bars represent 100 µM in subpanels A1-E1 and 50 µM in subpanels A2-E2. (E) Quasistatic lung compliance in DC-depleted CD11c-DTR mice that were injected (+) or not (−) with the indicated antibodies. All mice were analyzed 90 minutes after the second injection. For the statistical analyses, only significant comparisons of interest are shown. Panel C was analyzed with a 1-way ANOVA with a Tukey’s post hoc test, panel E was analyzed by a 1-tailed unpaired t test. Each dot represents 1 mouse, and error bars represent SD. ***P < .001, ****P < .0001.

CD4⁺CD25⁺FoxP3⁺Tregs protect against antibody-mediated acute lung injury. (A-B) Lung W/D weight ratios of untreated (−) BALB/c mice or mice that first underwent in vivo depletions of T cells (A) or B cells (B) by the indicated antibody injections (+) and were then injected with the TRALI-inducing antibody 34-1-2s or indicated control antibodies. (C) Representative flow cytometric dot plot analysis of spleen, thymus, peripheral blood, and lung cells showing the extent of a typical in vivo depletion of CD4⁺CD25⁺FoxP3⁺ Tregs (splenic cells were depleted from 9.82% to 2.4%, thymic cells were depleted from 0.59% to 0.1%, peripheral blood cells were depleted from 2.52% to 0.2%, and lung cells were depleted from 3.7% to 0.5% of total CD4⁺ T cells). (D) Representative macroscopic images of lungs from a Treg-depleted mouse 90 minutes after injection of the indicated control antibody (left) or 34-1-2s (right). (E) Lung W/D weight ratios of CD4⁺CD25⁺FoxP3⁺ Treg-depleted BALB/c mice injected with the indicated isotype control antibodies and/or 34-1-2s. (F) Lung histology from BALB/c mice receiving the indicated antibody injections: subpanels A1-E1 and A2-E2 represent lung tissue images taken at magnification ×20 and ×40, respectively. Representative images of each indicated group are shown. A zoom of indicated square in E2 is depicted alongside and shows alveolar PMN infiltration. Scale bars represent 100 µM in subpanels A1-E1 and 50 µM in subpanels A2-E2. (G) Measurement of tissue elastance in Treg-depleted BALB/c mice that received either 34-1-2s injection or indicated isotype control. All mice were analyzed 90 minutes after the second injections. For the statistical analyses, only significant comparisons of interest are shown. Panel A was analyzed with a 1-way ANOVA with a Tukey’s post hoc test, panel E was analyzed with a 1-way ANOVA with Dunn’s post hoc test, and panel G was analyzed by a 1-tailed Mann-Whitney test. Each dot represents 1 mouse, and error bars represent SD. *P < .05, ***P < .001, ****P < .0001. IgG, immunoglobulin G; NS, nonsignificant.