Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking

Abstract

Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3(+) (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19(+) B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.

Fig. 1

Effects of ACRS on presence of leukocytes in the blood. The absolute number of lymphocytes, monocytes and granulocytes were enumerated from 1 to 24 h following ACRS treatment. ACRS (1 h) mice and control mice were sacrificed immediately (1 h time point) or allowed to recover in their cages for varying amounts of time prior to bleeding. At early times (1 and 2 h), ACRS suppressed the numbers of lymphocytes and elevated the numbers of neutrophils

Fig. 2

β-AR antagonist effects on peripheral blood leukocyte numbers after ACRS. BALB/c mice treated IP with atenolol (a) or ICI 118,551 (b) 20 min prior to ACRS. At 2 h following the initiation of ACRS, blood was assayed for presence of leukocyte subsets by flow cytometry. Statistical analysis: one-way analysis of variance was used with either Tukey or Dunn’s post hoc test. The p (<0.05) value comparisons are as follows: (*) versus vehicle control group

Fig. 3

Peripheral blood leukocytes of BALB/c mice that were injected IP with vehicle (PBS), atenolol (20 mg/kg BW), or ICI 118,551 (15 mg/kg BW) 20 min prior to initiation of ACRS. At 2 h following the initiation of ACRS, a whole blood sample was taken for analysis by flow cytometry. Statistical analysis: one-way analysis of variance with Tukey post hoc test. The p (<0.05) value comparisons are as follows: a versus vehicle control group; b versus vehicle ACRS group; c versus treatment (atenolol or ICI-118,551) control group

Fig. 4

Bone marrow neutrophils were isolated and sorted based on GR-1⁺/CD11b⁺ staining by BD FACSAria sorting, which enriched the neutrophil population from 53 % to 91 % (a). This concentrated neutrophil population was used to determine β1-AR mRNA expression (b) in reference to BALB/c (lanes 2 & 4) and β1β2KO (lanes 3 & 5) neutrophil (lanes 2 & 3) and spleen (lanes 4 & 5) preparations. Lane 1 contained nucleotide bp control markers and lane 6 was a negative control. PCR was performed on the same mice to ensure they expressed the proper knock-out genes (c); lanes 1, 2 and 3 represent BALB/c cells, β1β2KO cells, and negative control, respectively

Fig. 5

Expression of β1-AR by peripheral blood leukocytes. After RBC lysis, cells were washed, fixed, and permeabilized. Based on flow cytometric separation of blood leukocytes with light scatter and antigens (see Materials and methods), the permeabilized subpopulations were assayed for the amount of β1-AR (Geomean of Fluroescence Intensity) with rabbit IgG or rabbit polyclonal antibodies to the intracellular cytoplasmic domain of β1-AR followed by FITC-goat anti-rabbit IgG

Fig. 6

ACRS induction of nitric oxide. Serum was deproteinized and the level of nitric oxide was assayed by conversion to nitrite as described in Methods. The sera were measured from 2 to 24 h after ACRS initiation; at 2 and 6 h, there were significant (*) increases in nitric oxide

Fig. 7

Leukocyte subsets from whole blood were measured at 2 h following the initiation of ACRS for GSH Levels. RBCs were removed (i.e., hypotonic lysis of RBCs) from the whole blood sample and washed thoroughly. The leukocytes were fixed with 2 % paraformaldehyde and permeabilized using Cytofix/Cytoperm. Permeabilized cells were incubated with 10 mM NEM for 20 min, washed and then analyzed by Flow cytometry using a fluorescently labeled 8.1 GSH-NEM antibody. ACRS induced no significant change in any glutathione levels

Fig. 8

BALB/c mice were split into two groups: control or ACRS. Mice in the ACRS group were subjected to ACRS for 1 h at 4 °C. Whole blood and bone marrow was harvested 24 h after the initiation of stress to evaluate leukocyte profiles; *p < 0.05

Fig. 9

BALB/c mice were split into two groups: control or ACRS. Mice in the ACRS group were subjected to ACRS for 1 h at 4 °C. Whole blood was immediately collected and allowed to clot for 30 min. The serum from the control and ACRS was removed and pooled for future analysis. All chemotaxis experiments used a chemotaxis media containing 5 % control or ACRS serum in a transwell chemotaxis system without (a, b) or with transwells pre-coated with 100 μl of 1 mg/ml Matrigel (c, d). There were no differences between control and ACRS sera

Fig. 10

BALB/c and β1-AR^−/− mice were split into two groups: control or ACRS. Mice in the ACRS group were subjected to ACRS for 1 h at 4 °C; 2 h after the initiation of ACRS, whole blood and bone marrow were harvested to determine leukocyte profiles by flow cytometry; *p < 0.05

Fig. 11

BALB/c or β1-AR^−/− mice were split into two groups: control or ACRS. Mice in the ACRS group were subjected to ACRS for 1 h at 4 °C. At 2 h following the initiation of ACRS, bone marrow was harvested and used to determine neutrophil chemotaxis to SDF-1α (100 ng/ml) using the modified Matrigel Transwell chemotaxis assay. There were no significant effects of ACRS or absence of β1-AR on chemotaxis