Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation

Abstract

Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. Here, we show that the process of neutrophil apoptosis is delayed in several inflammatory diseases, suggesting that this phenomenon may represent a general feature contributing to the development of neutrophilia, and, therefore, in many cases to host defense against infection. The delay of neutrophil apoptosis was associated with markedly reduced levels of Bax, a pro-apoptotic member of the Bcl-2 family. Such Bax-deficient cells were also observed upon stimulation of normal neutrophils with cytokines present at sites of neutrophilic inflammation, such as granulocyte and granulocyte-macrophage colony-stimulating factors, in vitro. Moreover, Bax-deficient neutrophils generated by using Bax antisense oligodeoxynucleotides demonstrated delayed apoptosis, providing direct evidence for a role of Bax as a pro-apoptotic molecule in these cells. Interestingly, the Bax gene was reexpressed in Bax-deficient neutrophils under conditions of cytokine withdrawal. Thus, both granulocyte expansion and the resolution of inflammation appear to be regulated by the expression of the Bax gene in neutrophils.

Figure 1

Delayed neutrophil death is a general feature of neutrophilic inflammation. Blood and BAL neutrophils from adult patients with three different inflammatory lung diseases (cystic fibrosis, n = 13; pneumonia, n = 1; and idiopathic pulmonary fibrosis, n = 2) associated with neutrophilia were cultured in the presence and absence of the neutrophil survival factor GM-CSF for the indicated times before cell death was measured by flow cytometry. The results were compared with blood neutrophils from normal control individuals (n = 10) and cancer patients with associated blood neutrophilia (n = 2). Values are means ± SEM (SEM is mostly concealed by the symbols). n.d., not done.

Figure 2

Bax levels are reduced in neutrophils under inflammatory conditions. (A) Flow cytometry. Freshly purified normal blood neutrophils expressed significant amounts of Bcl-x_L and Bax, but no Bcl-2 or Bcl-x_S protein. In contrast to normal neutrophils, inflammatory diseases neutrophils (cystic fibrosis and cancer with associated neutrophilia, see also Fig. 6B) expressed little or no detectable Bax protein. (B) The markedly reduced Bax expression in inflammatory diseases neutrophils (cystic fibrosis) was confirmed by immunoblotting. (C) Semiquantitative RT-PCR. Bax protein expression correlates with the expression of Bax mRNA. Inflammatory diseases neutrophils (cystic fibrosis) had markedly reduced Bax mRNA levels. Numbers of PCR cycles are given at the top. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control amplifications were performed demonstrating equal quality of the cDNA preparations derived from blood, BAL, and sputum neutrophils (not presented). Data are representative of at least five independent experiments.

Figure 3

Bax-deficient neutrophils can be generated by exposure of normal neutrophils to survival factors in vitro. (A) Flow cytometry. Normal neutrophils were cultured in the presence or absence of cytokines and LPS. (B) The effects of GM-CSF and G-CSF on Bax protein levels in normal neutrophils were confirmed by immunoblotting. (C) RT-PCR. GM-CSF exposure of normal neutrophils rapidly reduced Bax mRNA levels. Data from A–C are representative of at least five independent experiments. (D) Cell death assay. Neutrophils were cultured in the presence or absence of cytokines and LPS, respectively, for 72 h before cell death was measured by flow cytometry. Values are means ± SEM of six independent experiments (*, P < 0.05).

Figure 4

Bax antisense (as Bax) but not scrambled oligodeoxynucleotides (sc Bax) reduce Bax protein expression in neutrophils. (A) Flow cytometry. Control and inflammatory diseases neutrophils (cystic fibrosis) were cultured in the presence of as Bax or sc Bax for 48 h. (B) Immunocytochemistry. (×1,000.) Control neutrophils were cultured in the presence of GM-CSF, sc Bax, and as Bax for 48 h. Data are representative of at least three independent experiments.

Figure 5

Bax antisense (as Bax) but not scrambled oligodeoxynucleotides (sc Bax) block neutrophil apoptosis in vitro. (A) Cell death assay. Control neutrophils were cultured at the indicated conditions for 48 h before cell death was measured by flow cytometry. (Right) DNA fragmentation (black) was markedly reduced in as Bax-treated compared to sc Bax-treated neutrophils, suggesting delayed apoptosis in Bax-deficient cells. (B) Cell death assay. Control neutrophils were pretreated with GM-CSF for 32 h to reduce Bax levels (see Fig. 3A). Bax-deficient neutrophils were then cultured under the indicated conditions for an additional 48 h before cell death was measured by flow cytometry. (Right) DNA fragmentation (black) is markedly reduced in as Bax-treated compared to sc Bax-treated neutrophils, suggesting delayed apoptosis in neutrophils where Bax reexpression was prevented. Values in the cell death assays are means ± SEM of three independent experiments (*, P < 0.05).

Figure 6

G-CSF and GM-CSF are significantly expressed in inflammatory responses associated with neutrophilia in vivo. (A) Immunohistochemical staining of cystic fibrosis lung tissues with the indicated antibodies. (×400.) G-CSF and GM-CSF were highly expressed in epithelial cells, macrophages, and neutrophils in patients with cystic fibrosis. (Upper) Central lung sections. (Lower) Lung alveoli. (B) Staining of tissue infiltrated by hypopharynx cancer cells. (×400.) The cancer cells demonstrated high G-CSF and GM-CSF expression. As a consequence, the patient developed a neutrophilia (during the time of these investigations, the peripheral blood neutrophil numbers were between 49,920 and 57,134 per μl of blood).

Figure 7

Bax-deficient neutrophils activate the Bax gene under conditions of cytokine withdrawal. (A) Freshly purified Bax-deficient inflammatory diseases neutrophils (0; cystic fibrosis, see Fig. 2A) were cultured without cytokine support for the indicated times, and Bax protein expression was determined by flow cytometry. (B) Freshly purified normal neutrophils (0) were cultured in the presence of G-CSF for 30 h to down-regulate intracellular Bax protein levels (see Fig. 3A). Data are representative of five independent experiments.