SerpinB1 protects the mature neutrophil reserve in the bone marrow

Abstract

SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE, CG, and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes, including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage, coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury, neutrophils were recruited to the lungs, causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow, coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature, postmitotic neutrophils. Finally, upon overnight culture, apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively, these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses.

Figure 1.. Reduced bone marrow PMN reserve in serpinB1^−/− mice.

(A) Representative flow cytometry plots of bone marrow cells of WT (+/+) and serpinB1^−/− (−/−) mice showing the gating strategy. Identified leukocyte (CD45⁺) subsets are mature neutrophils (PMNs; CD11b⁺Ly-6G⁺, red), myelocytes (Myelo; CD11b⁺Ly-6G^negSSC^hiCD115^neg, orange), promonocytes/monocytes (Mono; CD115⁺, green), and B cells (CD19⁺, blue). Percentage (B) and absolute count (C) for each bone marrow subset and lineage-negative cells (lin^neg; CD45⁺CD11b^negLy-6G^negCD19^negCD115^neg) are shown. WBC, White blood cell.

Figure 2.. Depletion of the bone marrow PMN reserve in lung injury.

PMN numbers in bone marrow (A) and BAL (B) 24 h after intranasal instillation of LPS or PBS vehicle for control (CTRL) mice (mean±sem; n=6–10/group from two independent experiments).

Figure 3.. SerpinB1 expression and function in myeloid progenitors.

(A) Representative flow cytometry plot of human bone marrow cells with subset gating based on SSC and CD45 expression [25, 26]. The black-dotted gate includes hematopoietic stem/progenitors and myeloblast cells. The latter differentiate into the neutrophil lineage (red arrow) and successively, promyelocytes (brown), myelocytes/metamyelocytes (orange), and band/mature neutrophils (red). Other lineages are also identified as lymphoid (blue) and monocytic (green). (B) MFI of gated subsets after intracellular staining for SERPINB1 (n=6). MFI for isotype control was <10. ProM, promyelocyte; Myel/MetaM, myelocyte/metamyelocyte; Mono, monocyte. (C) Frequency of CFUs of multipotential [granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM)], granulocyte/macrophage (GM), granulocyte (G), and macrophage (M) progenitors in WT (+/+) and serpinB1^−/− (−/−) bone marrow (mean±sem; n=3). (D) CFU counts/2 × 10⁴ bone marrow (BM) cells. Mean of three independent experiments is shown. (E) Serum G-CSF concentrations in WT (+/+) and serpinB1^−/− (−/−) mice.

Figure 4.. Myelocyte proliferation and granulopoiesis.

Percentage (A) and absolute numbers (B) of Ki-67⁺ cells among bone marrow neutrophils (CD11b⁺Ly-6G⁺), myelocytes (CD11b⁺Ly-6G^negSSC^hi), and promonocytes/monocytes (CD115⁺) of WT (+/+) and serpinB1^−/− (−/−) mice. (C) Absolute numbers of EdU⁺ PMNs in the bone marrow. (D) Absolute numbers of EdU⁺ myeloid/monocytic precursors (MP; CD11b⁺Ly-6G^neg; including myelocytes and CD11b⁺ monocytes) in bone marrow. Absolute numbers of EdU⁺ blood PMNs (CD11b⁺Ly-6G⁺) (E) and monocytes (CD11b⁺Ly-6G^neg) (F) in the same animals. Pooled data from two independent experiments (mean±sem; n=4/time-point; **P<0.01; ***P<0.001). For all panels, WT and serpinB1^−/− are shown as black and white symbols, respectively.

Figure 5.. Reduced survival of serpinB1^−/− bone marrow PMNs.

Survival of isolated bone marrow PMNs after 16 h culture in vitro was assessed by simultaneous staining with Ly-6G, 7-AAD, and annexin V-FITC. Apoptotic neutrophils (A) are reported as annexin V-positive within Ly-6G⁺ cells with normal (high) FSC and SSC. Necrotic neutrophils (B) are reported as 7-AAD⁺ within Ly6G⁺ cells including FSC^low cells. Data were analyzed by paired t-test (mean±sem; n=5). (C) Western blot analysis of FACS-sorted bone marrow PMN lysates stained with indicated antibodies. SerpinB1 is found as 42 kDa-native (closed arrowhead), 66 kDa-complex with neutrophil proteases (open arrowhead), and 38 kDa-post-complex (*) forms. Data are representative of two independent preparations.

Figure 6.. CY myelosuppression and stress granulopoiesis.

(A) Absolute numbers and (B) percent PMNs in peripheral blood before and after CY injection. Serum G-CSF (C) and bone marrow fluid NE activity (D) were measured at indicated days after CY injection. (limit of detection for NE was 8 ng/ml). Data were pooled from two independent experiments (mean±sem; n=4–8/time-point).