Acute stress transiently activates macrophages and chemokines in cervical lymph nodes

Forty male Sprague–Dawley rats (10–12 weeks old, body weight 432 ± 30 g; SLC Japan, Hamamatsu, Japan) were used in this study. Experiments were conducted from December 2019 to August 2021. All animals were housed in an experimental animal facility, where the managers monitored animal health and behavior twice per day. The animals were maintained in a temperature-controlled room (23 ± 3 °C) with a 12/12 h light/dark cycle (lights were switched on at 7:00 h). Two to three rats were housed per cage (made of polypropylene; dimensions: 307 mm wide, 405 mm deep, and 227 mm high), and the rats had free access to water and food. The animals were treated humanely, taking all due measures to alleviate suffering. The Review/Ethics Committee of Jichi Medical University approved all experimental procedures, which were conducted according to the University Guidelines for Animal Experimentation and National Institutes of Health Guide for the Care and Use of Laboratory Animals (December 2019 Approval number: 18038-03).

The animals were divided randomly into four groups (n = 6): a control group (C) and three experimental groups subjected to restraint stress (RS) for 30 min (RS30), 60 min (RS60), and 120 min (RS120). Animals were tied to a wooden board for 30, 60, and 120 min, respectively, to induce RS. Leg fasteners held the rats in the supine position (spread-eagle position). All stress induction procedures and euthanasia were performed between 11:00 and 17:00 h. RS was induced as described in our previous reports [17, 18, 22].

Immediately after each stress induction time was over, the animals were transferred into an airtight plastic case, anesthetized using inhalational anesthesia with 5% halothane (2-bromo-2-chloro-1,1,1-trifluoroethane; Takeda Chemical Industries, Osaka, Japan), and euthanized by intraperitoneal overdose of sodium pentobarbital (85 mg/kg; FUJIFILM Wako Chemical Corp., Osaka, Japan). The death of animals was confirmed by checking for whole body atony and respiratory arrest. After decapitation, the cervical lymph nodes were harvested from each group for microarray and reverse transcription-quantitative PCR (RT-qPCR) analyses. The organs were collected into plastic tubes and stored at −80 °C until analysis.

The total RNA was extracted from the cervical lymph nodes of the four groups using ISOGEN reagent (Nippon Gene Co. Ltd., Toyama, Japan), according to the manufacturer’s instructions. The total RNA concentration and purity were measured spectrophotometrically using a NanoDrop1000 (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was monitored by electrophoresis on an Agilent Bioanalyzer RNA 6000 Nano (Agilent Technologies, Santa Clara, CA, USA).

The extracted RNA (100 ng) was subjected to Cy3-labeled cRNA synthesis using a Low Input Quick Amp Labeling Kit (5190-2305: Agilent Technologies). The amplified RNA and dye incorporation were quantified using the Agilent Bioanalyzer RNA 6000 Nano and hybridized to a microarray chip (SurePrint G3 Rat GE Microarray 8X60K ver. 2.0, Agilent Technologies). After hybridization, the chips were washed using the Gene Expression Wash Pack. The arrays were scanned with an Agilent SureScan G4900DA, and fluorescence intensity was extracted using Feature Extraction software ver. 11.5.1.1.

Raw data of the microarray intensities were normalized using the 75th percentile shift method with GeneSpring software ver. 14.9 (Agilent Technologies) for inter-microarray variability. Differential gene expression was defined as a two-fold change relative to that of the control. We performed Gene Ontology (GO) analysis to summarize the biological functions of the selected genes. The data were then processed using Fisher’s exact test and multiple test correction to identify significant over-representation of GO annotations belonging to the selected genes.

The total RNA was extracted using the ISOGEN reagent (Nippon Gene, #311-07361), as described above, from the four groups. These concentrations were determined using the NanoDrop1000. cDNA was synthesized using a Transcriptor First-Strand cDNA synthesis kit (#04897030001, Roche Diagnostics Ltd., Basel, Switzerland), following the manufacturer’s protocol. Briefly, the template–primer mixture was denatured by heating the tube for 10 min at 65 °C in a heated block. The tube was briefly centrifuged and immediately cooled on ice. Master mix was added, and the tube was placed in a thermal cycler with a heated lid for 60 min at 50 °C. Reverse transcriptase was inactivated by heating the tube to 85 °C for 5 min, and the samples were chilled on ice. The synthesized cDNA stock was stored at −25 °C until analysis. qPCR was performed on a LightCycler® TaqMan Master (#04735536001, Roche Diagnostics Ltd.) using commercially available TaqMan probes with a dye label (FAM) on the 5′ end and a minor groove binder and a non-fluorescent quencher on the 3′ end, CXCL1 (Rn00578225_m1) [23], CXCL2(Rn00586403_m1) [24, 25], CXCR2(Rn02130551_s1) [24, 26], and GAPDH(Rn01775763_g1) [27] (Thermo Fisher Scientific, Waltham, MA, USA). The thermal cycling conditions provided for the LightCycler® 2.0 System were as follows: enzyme activation: 95 °C for 10 min, 45 cycles of amplification: 95 °C for 10 s, 60 °C for 30 s, and signal detection at 72 °C for 1 s, and cooling at 40 °C for 30 s. Gapdh was used as the reference gene to normalize the expression levels.

We performed a histological examination to identify the tissues exhibiting increased Cxcl1, Cxcl2, and Cxcr2 mRNA levels in the C and RS30 groups. For immunohistochemistry, animals (n = 3 in each group) were anesthetized and euthanized as described above. The animals were then transcardially perfused with 0.85% NaCl followed by 4% formaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer (PB; pH 6.9). The cervical lymph nodes were dissected and fixed in the same fixative for 24 h. After immersing the samples in 20% sucrose, the lymph nodes were cut into 10-μm-thick sections using a sliding microtome (Yamato Kohki Industrial Co. Ltd., Asaka, Japan) equipped with a frozen stage. The sections were stored in 0.1 M PB (pH 7.4) containing 0.9% phosphate-buffered saline (PBS) until immunostaining. Some sections were stained with hematoxylin and eosin to examine the general histology. Immunohistochemistry was performed as described in our previous report [22]. Briefly, the sections were washed overnight and incubated with the following primary antibodies: mouse anti-CD68 antibody (ab31630; Abcam, Cambridge, UK; 1:100), rabbit anti-CXCL1 polyclonal antibody (bs-10234R, Bioss Antibodies, Boston, MA, USA; 1:500), rabbit anti-CXCL2 polyclonal antibody (LS-C294372; LifeSpan BioSciences, Inc., Seattle, WA, USA; 1:500), and rabbit anti-CXCR2 polyclonal antibody (orb1720; Biorbyt Ltd., Cambridge, UK; 1:500). After washing, the sections were incubated with biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories, Burlingame, CA, USA; 1:100) for 1 h. For CD68, the sections were incubated with biotinylated donkey anti-mouse IgG (Millipore Corporation, Billerica, MA, USA; 1:100). The sections were washed and incubated for 30 min at room temperature with avidin–biotin–horseradish peroxidase complex (PK-6100; Vector Laboratories; 1:200). After the final wash, the sections were reacted with 0.02% 3,3′-deaminobenzidine tetrahydrochloride and 0.005% hydrogen peroxide in 0.05 M Tris-HCl (pH 7.4), counterstained with thionine, and coverslipped using Malinol (Muto Pure Chemicals Co. Ltd., Tokyo, Japan). For double staining of CD68 and CXCR2, the samples were treated with Alexa Fluor 488-conjugated donkey anti-mouse IgG specific for CD68 (ab150105; Abcam; 1:100) and Texas red–conjugated donkey anti-rabbit IgG specific for CXCR2 (GTX26800; GeneTex, Inc., Irvine, CA, USA; 1:100). PBS was used for all washing steps, and PBS containing 1% bovine serum albumin and 0.3% Triton X was used to dilute the antibodies. The cells were counted using printed images, and the areas with cells were calculated from the weights of printed papers.

In situ hybridization (ISH) was performed according to the manufacturer’s protocol (GenoStaff Co., Ltd., Tokyo, Japan). Animals (n = 3 in each group) were sacrificed as described above, except that 4% formaldehyde in PB (pH 7.4) was used as the fixative. The cervical lymph nodes were collected and embedded in paraffin wax after dehydration. Next, 6-μm-thick sections were mounted on MAS-coated glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan). The sections were dewaxed using xylene and then hydrated in decreasing ethanol concentrations followed by distilled water. The sections were treated with 0.2% hydrochloric acid and 5 μg/mL protease K (FUJIFILM Wako Chemical Corp.) at 37 °C for 10 min each. After washing with PBS and hybridization buffer, the sections were hybridized to relevant probes at 60 °C overnight. Digoxigenin-labeled antisense and sense RNA probes were synthesized by GenoStaff Co., Ltd. (for rat CXCL1, sequence position: 636–864, size: 229 bases; for rat CXCL2, sequence position: 727–1047, size: 321 bases; for rat CXCR2, sequence position: 16–266, size: 251 bases). After washing with hybridization buffer, the sections were washed with 50% formamide for 10 min at 60 °C. The sections were washed with hybridization buffer and then with Tris-HCl buffer (pH 7.0) containing 0.8% NaCl, 0.02% KCl, and 0.2% Tween-20 at room temperature. Digoxigenin was detected with alkaline phosphatase-labeled anti-digoxigenin Fab fragments (32871920; Roche Diagnostics, Mannheim, Germany; 1:1,000 dilution), and signals were visualized using a mixture of nitrotetrazolium blue chloride (Sigma–Aldrich, St. Louis, MO, USA) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Sigma–Aldrich) in Tris-HCl buffer (pH 9.5). The sections were counterstained with Kernechtrot staining solution (Muto Pure Chemicals Co., Ltd.) and coverslipped with Malinol.

Data are presented as the mean ± standard error of the mean. Differences between groups were analyzed by one-way analysis of variance, followed by Tukey’s post-hoc test. Results with P < 0.05 were considered statistically significant. Immunohistochemical and in situ histochemical data were statistically analyzed using t-tests. Statistical analysis was performed using Statcel4 software (OMS Publishing, Inc., Saitama, Japan).

We screened a total of 26,186 genes using microarray analysis. Among them, the expression of 514 and 496 genes was upregulated and downregulated, respectively, by 2-fold or more in the RS30 group compared with that in the C group. Notably, we observed 23- and 13-fold higher expression levels of Cxcl1 and Cxcl2, respectively, in the RS30 group than in the C group. Furthermore, the expression of Cxcr2 was upregulated by approximately 1.6-folds in the RS30 group compared with that in the C group (Fig. 1a). Using a database (Gene Ontology Consortium (http://​www.​geneontology.​org)), microarray analysis data were integrated to identify the GO terms and biological processes related to the three upregulated genes. The top 15 significantly enriched GO terms associated with the three upregulated genes are presented in Fig. 1b.

Stress immediately induced transient upregulation of Cxcl1/2 mRNA expression in the RS30 group (P < 0.01 compared with the C group based on a Tukey’s post-hoc test). Notably, the expression levels were significantly downregulated in the RS60 and RS120 groups (P < 0.01) compared with the expression levels in the RS30 group, and the levels recovered to the normal levels in the C group. In contrast, the mRNA levels of Cxcr2 were significantly increased in the RS30 and RS120 groups (P < 0.05) compared with that in the C group. The mRNA levels tended to remain at higher levels in the RS60 group than in the C group, although the differences were not significant (Fig. 2).

Cervical lymph nodes in the RS30 group showed frequent erythrocyte accumulation in the medullary sinus; this accumulation was not observed in the C group (Fig. 3a, b). The accumulated erythrocytes also formed rosettes enclosing macrophages (Fig. 3c, d). Erythrocyte accumulation was rarely observed in the RS60 group and was not detectable in the RS120 group.

CD68-immunoreactive macrophages were mainly localized in the medullary sinus (Fig. 4a-e). In the cervical lymph nodes of the C group, CD68-immunoreactive macrophages were slender and scattered (Fig. 4a, b). In contrast, the macrophages in the RS30 group were swollen and more densely located in the lymph nodes (Fig. 4c, d). The number of CD68-immunoreactive cells (per 100 μm²) in the medullary sinus was 14.3 ± 2.9 and 18.8 ± 5.0 in the C and RS30 groups, respectively (P < 0.01) (Fig. 4e).

CXCL1-immunoreactive cells were mainly localized in the medullary cords where B/plasma cells were present (Fig. 5a-d). Immunoreactive puncta were eccentrically localized to the cell nucleus (Fig. 5b, d), presumably corresponding to cisternae of the rough endoplasmic reticulum and/or Golgi complex of plasma cells. In some presumed B cells, immunoreactivity was extended to the whole cell bodies (Fig. 5b, d). Based on the cell size (7–9 μm in diameter), paucity of the cytoplasm, and location in the B/plasma cell territories, these immunoreactive cells were predicted to be B cells. The number of these cells was smaller than that of immunoreactive puncta-bearing cells. The number of immunoreactive puncta per 100 μm² was 78.8 ± 17.3 and 100.8 ± 16.6 in the C and RS30 groups, respectively (P < 0.05) (Fig. 6a). Concomitantly, ISH signals were observed in areas eccentrical to the cell nucleus (Fig. 5e, f). The number of B/plasma cells with these features was larger in the RS30 group (Fig. 5f) than in the C group (Fig. 5e). The number of Cxcl1 ISH-positive cells per 100 μm² was 89.8 ± 33.2 and 129.2 ± 23.5 in the C and RS30 groups, respectively (P < 0.05) (Fig. 6b).

Immunohistochemically, CXCL2-immunoreactivity was observed in endothelial cells of the high endothelial venules in the cortex (Fig. 7a–d) but not in those of the ordinal venules in the cortex and medulla. Furthermore, the immunoreactive intensity increased in endothelial cells of the high endothelial venules in the RS30 group (Fig. 7c, d). The percentages of immunopositive areas against the entire endothelial cell area of the high endothelial venules were 42.9 ± 12.2% in the C group and 64.2 ± 13.8% in the RS30 group (P < 0.05) (Fig. 8a). Additionally, the ISH findings agreed with those of the immunohistochemical analysis (Fig. 7e, f). Although the positive signals in the cervical lymph nodes were not prominent in the C group (Fig. 7e), they increased significantly in the RS30 group (Fig. 7f). The percentages of Cxcl2 ISH-positive cell numbers compared to the total number of cells in the high endothelial venules were 29.3 ± 6.1% and 57.2 ± 13.2% in the C and RS30 groups, respectively (P < 0.05) (Fig. 8b).

Furthermore, CXCR2-immunoreactive cells were mainly observed in the medullary sinus (Fig. 9a–d). In the C group, the cells were slender and scattered (Fig. 9a, b); in stressed animals, these cells were swollen and denser (Fig. 9c, d). Double staining for CD68 and CXCR2 indicated that CXCR2-immunoreactive cells corresponded to CD68-immunoreactive macrophages (Fig. 9e, f). Furthermore, ISH revealed intense signals in macrophages from the RS30 group (Fig. 10b) compared with those from the C group (Fig. 10a). In addition, Cxcr2 signals were observed in the medullary lymphocytes, showing that the intensity was higher in the RS30 group (Fig. 10b) than in the C group (Fig. 10a). The number of Cxcr2 ISH-positive cells per 100 μm² was 64.7 ± 20.6 and 145.1 ± 34.1 in the C and RS30 groups, respectively (P < 0.01) (Fig. 10c).