The intriguing normal acute inflammatory response in mice lacking vimentin

Abstract

Neutrophils express only two intermediate filament proteins, vimentin and, to a lesser extent, lamin B. Lamin B mutant mice die shortly after birth; however, mice lacking vimentin (vim(-/-)) develop and reproduce normally. Herein, we investigate for the first time the role of vimentin in general inflammation in vivo and in neutrophil functions ex vivo. Using the murine air pouch model, we show that the inflammatory response induced by lipopolysaccharide, interleukin-21 or carageenan is, intriguingly, uncompromised in vim(-/-) mice and that neutrophil functions are not altered ex vivo. Our results suggest that vimentin is dispensable for the establishment of an acute inflammatory response in vivo. In addition, based on several criteria presented in this study, one has to accept the existence of a very complex compensatory mechanism to explain the intriguing normal inflammatory response in absence of vimentin.

Fig. 1

Role of vimentin during an inflammatory response induced by lipopolysaccharide (LPS) in vivo. Dorsal air pouches were raised in vim^+/+, vim^+/− and vim^−/− mice as described in Materials and methods before injecting 1 ml of the buffer (Ctrl) or LPS (1 µg/ml) directly into the pouch. Six hours later, the exudates were harvested and the number of emigrated total leucocytes was calculated (a) and separated into granulocytes (open bar) or monocytic cells (solid bar) (b, c). (a) Results are means ± standard error of the mean (s.e.m.) (n ≥ 14, males + females, five different experiments). (b) Cell identification was performed by cytology using the Hema-stain staining kit. Inset, a representative cytocentrifuged preparation of harvested cells illustrating that the majority of cells attracted by LPS are granulocytes in both vim^−/− and vim^+/+ mice. C, cells were stained with purified rat anti-mouse 7/4 monoclonal antibody directed against murine neutrophils (open bar) or rat anti-mouse F4/80 antigen antibody recognizing murine monocyte/macrophages (solid bar) and analysed by flow cytometry as described in Materials and methods. Inset, a representative data plotted in the bar graph (the appropriate isotypic controls are illustrated in grey). (b, c) Results are means ± s.e.m. (n = 6 animals, three different experiments). No significant differences were observed between vim^+/+, vim^+/− and vim^−/− regarding the number of cells attracted as well as the percentages and subtype of cell populations (analysis of variance).

Fig. 2

Evidence that the protein pattern expressions are identical in vim^–/– and vim^+/+ neutrophils. (a) Mice genotyping was performed systematically by polymerase chain reaction using the three different primers, as described in Materials and methods according to Eckes et al. [8]. The 1–2 primer pair allows amplification of a 398-base pairs (bp) fragment when the wild-type allele is present and the 2–3 primer pair allows amplification of a 530-bp fragment when the disrupted allele is present [8]. (b) Expression of the cytoskeletal proteins vimentin, lamin B₁ and vinculin in harvested neutrophils following lipopolysaccharide (LPS)-induced murine air pouch. Results are representative of at least 23 animals (total). (c) Exudates were collected from the pouch and separated into two fractions: cells pellet (not shown) and supernatants. Proteins from were run by electrophoresis and gels were stained with silver staining for extracellular proteins (5–20% sodium dodecyl sulphate–polyacrylamide gel electrophoresis). Molecular weights are indicated on the left side. (b, c) Results are representative of at least 15 or 18 different animals, respectively. (d) Antibody array assay was performed with exudates collected and pooled from 13 LPS-induced air pouches for wild-type and knock-out mice (for a total of 4·6 ± 0·8 and 4·8 ± 0·9 leucocytes attracted/pouch, respectively). The cytokines/chemokines that are the most affected by LPS are boxed. Note the presence of two dots per analyte, as all cytokines/chemokines were tested in duplicate. No differences were noted when the intensity of each spot was compared with the corresponding positive control. The list of all cytokines/chemokines is detailed in the Materials and methods.

Fig. 3

Mice lacking vimentin possess the same number of leucocytes as well as the same proportions of lymphocyte, monocytic and neutrophil cells. (a) Blood collection was performed by cardiac puncture from vim^+/+ (wild-type) or vim^−/− (knock-out) animals and leucocyte counts were assessed by cytology as described in Materials and methods. (b) Differential cell count was performed with an aliquot of 0·5 × 10⁶ leucocytes cytocentrifuged on microscope slides and coloured with the Hema-3 stain set in order to differentiate lymphocytes (Lympho), monocytes-macrophages (Mono-mac) and neutrophils (neutro). Results are means ± standard error of the mean (n = 10 mice/group, three separate experiments). Inset, note the preponderance of murine lymphocytes (L) in the blood as opposed to neutrophils (N) or monocytes–macrophage (M), as expected [24].

Fig. 4

Absence of vimentin does not alter neutrophil and monocytic cell influx in vivo. (a, b) Dorsal air pouches were raised in vim^+/+ and vim^−/− mice before injecting 1 ml of the buffer (Ctrl), lipopolysaccharide (LPS) (1 µg/ml) or interleukin (IL)-21 (100 ng/ml) directly into the pouch. Six hours later, the exudates were harvested and the number of emigrated total leucocytes was calculated (a). Results are means ± standard error of the mean (s.e.m.) (n = 8, two separate experiments) and include males and females as no sexual dimorphism was observed. Differential counts were assessed by cytology (b, n ≥ 4 mice/group, pool of two experiments). (c, d) Dorsal air pouches were raised in vim^+/+ (wild-type) and vim^−/− (knock-out) mice before injecting 1 ml of the buffer (Ctrl), LPS (1 µg/ml) or carrageenan (CRGN, 1% in Hank's balanced salt solution) directly into the air pouch. Twenty-four hours later, the exudates were harvested and the number of emigrated total leucocytes was calculated (c). Results are means ± s.e.m. (n = 3 mice/group, two separate experiments) and include males and females. Differential counts were assessed by cytology (d). No significant differences were observed between wild-type and knock-out mice regarding the number of cells attracted as well as the percentages and subtype of cell populations (analysis of variance).

Fig. 5

The spontaneous apoptotic rate and its modulation by pro- or anti-apoptotic agents are not altered in neutrophils devoid of vimentin ex vivo. Neutrophils from vim^+/+, vim^+/− and vim^−/− mice were harvested after lipopolysaccharide (LPS)-induced air pouch and incubated in vitro for 22 h in the presence of buffer (Ctrl), the pro-apoptotic agents arsenic trioxide (AT, 5 µM) or Viscum album agglutinin-I (VAA-I, 1000 ng/ml) or the anti-apoptotic cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) (GM, 65 ng/ml) and apoptosis was assessed by flow cytometry by evaluating the number of fluorescein isothiocyanate (FITC)-annexin-V positive cells. (a) Results are means ± standard error of the mean (n = 10 mice/group, four separate experiments) and (b) (n = 5 mice/group, two separate experiments). Inset, representative experiment plotted in the bar graph. Arrows, illustrates the apoptotic cell population. *P < 0·05 versus control (Ctrl) by analysis of variance. No significant differences were observed between wild-type and knock-out mice.

Fig. 6

Role of vimentin in intracellular calcium mobilization, reactive oxygen species (ROS) generation and phagocytosis. Neutrophils from vim^+/+ (wild-type) and vim^−/− (knock-out) mice were harvested from lipopolysaccharide (LPS)-induced air pouches and adjusted at a concentration of 5 × 10⁶ cells/ml, before being incubated for calcium measurements as detailed in Materials and methods. The calcium ionophore A23187 was used at a final concentration of 10 µM. Phosphate-buffered saline (PBS) was used as a negative control. Inset, representative data plotted in the bar graph. Results are means ± standard error of the mean (s.e.m.) (n = 7, total). (b) Harvested cells were incubated in vitro in the presence or absence (Ctrl) of phorbol 12-myristate 13-acetate (PMA) (10⁻⁷ M) for the indicated periods of time. Mitochondrial ROS production was assessed by flow cytometry using DHR123. Results are means ± s.e.m. (n = 13 mice/group, three separate experiments). (c) Neutrophils from vim^+/+ (wild-type) and vim^−/− (knock-out) mice were harvested from LPS-induced air pouches and incubated in vitro in the presence of buffer (Ctrl), interleukin (IL)-4 (100 ng/ml), granulocyte–macrophage colony-stimulating factor (GM-CSF) (65 ng/ml, GM) or PMA (30 ng/ml) and phagocytosis was assessed by by counting the number of cells ingesting at least one opsonized-sheep red blood cell (SRBC). Results are means ± s.e.m. (n = 3 mice/group, four separate experiments) and are expressed as percentage of phagocytosis. Inset, vim^−/− neutrophils ingesting opsonized SRBC (arrows). *P < 0·05 versus control (Ctrl) by analysis of variance. No significant differences were observed between wild-type and knock-out neutrophils.