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. 2013 Feb 11;210(2):375-87.
doi: 10.1084/jem.20122183. Epub 2013 Jan 21.

Neutrophils Control the Magnitude and Spread of the Immune Response in a Thromboxane A2-mediated Process

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Free PMC article

Neutrophils Control the Magnitude and Spread of the Immune Response in a Thromboxane A2-mediated Process

Chiao-Wen Yang et al. J Exp Med. .
Free PMC article

Abstract

Neutrophils are obligate cells entering lymph nodes shortly after immunization with protein antigens in adjuvants, starting during the first hour and continuing for several days in two distinct waves. Previously, we demonstrated the strong suppressive effects of neutrophils on CD4 T cell and B cell responses, using either neutrophil-depleting antibodies or genetically neutropenic mice. In this study, we find that neutrophils are the major cells controlling the spread of T cell responses to distal lymph nodes. Although in the presence of neutrophils, ∼75% of the response was restricted to the draining node, in their absence, most of the response was found in distal nodes. Prostanoids were responsible for the rapid entry of neutrophils into the draining nodes, as well as for the two distinct neutrophil effects: the modulation of the magnitude of the cellular response, and in its spread outside the draining nodes. Neutrophil-produced thromboxane A(2) was the key eicosanoid controlling both effects. Adoptive transfer of neutrophils into mice genetically deficient in neutrophils indicated their role in both. These functions of neutrophils are important in infections and vaccinations with adjuvants where neutrophils are abundant in the initial stages.

Figures

Figure 1.
Figure 1.
Entry of two waves of neutrophils in the lymph nodes after immunization. (A) B10.BR mice were immunized with 10 nmol HEL/CFA. The numbers of neutrophils in the popliteal nodes at the indicated time after immunization were analyzed by FACS of CD11b+ Ly-6G+ cells and (B) immunofluorescence and H&E staining. Original magnification in B, ×40 (left); ×4 (middle); ×100 (right). Bars: 200 µm (left); 20 µm (middle); 500 µm (right). (C and D) B10.BR mice were i.p. injected with PBS, 40 µg/mouse indomethacin (INDO), or 500 ng/mouse of PTX 1 d before immunization (C); or 6 d after immunization (D). The number of neutrophils in the popliteal nodes was analyzed by FACS of CD11b+ Ly-6G+ cells at 2 h (C) or 7 d (D) after immunization. (E) B10.BR mice were i.p. injected with indomethacin at 1, 2, or 3 d before immunization. PBS was given 1 d before immunization. The number of neutrophils in the popliteal lymph nodes was analyzed after 2 h. (F) B10.BR mice were given PBS or indomethacin 1 d before immunization. The entry of wave 1 or wave 2 neutrophils in the popliteal nodes was analyzed by FACS of CD11b+ Ly-6G+ cells at 2 h (W1) or 6 d (W2) after immunization. (G and H) C57BL/6 wild-type, COX-1−/−, or COX-2−/− mice were immunized with 10 nmol OVA/CFA and the number of neutrophils in the popliteal nodes was analyzed by FACS at 2 h (G) or 7 d (H) after immunization. (A and B) Shown are representative data from 3 individual experiments with n = 2 in each group; (C–H) pooled results from 2–4 experiments, in which each dot presents data from individual mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2.
Figure 2.
Spread of T cell responses to distal draining lymph nodes in the absence of neutrophils. (A) B10.BR mice were i.p. injected with either neutrophil-depleting antibodies (1A8 aPMN; 1 mg/mouse) or control Ig 1 d before immunization with 10 nmol HEL/CFA. T cell recall responses in individual draining lymph nodes were measured by IL-2 and IFN-γ ELISPOT at day 7 after immunization. Shown are data from 3 individual experiments with n = 2 in each group. (B) Pooled results from 10 individual experiments. T cell recall responses by ELISPOT at 7 d after immunization were shown as percentage of total responses analyzed by the number of cytokine-producing cells in popliteal nodes or distal draining lymph nodes, including iliac, inguinal, renal, and axillary nodes. (C and D) CD4 or CD8 T cell recall responses in OVA/CFA immunization in C57BL/6 mice (C); or HEL/CFA immunization in NOD mice (D) were measured at day 7 after immunization by ELISPOT using CD4 or CD8 recall epitopes. (E) C57BL/6 wild-type or G-CSFR−/− mice were i.p. injected with 1A8 Ab or control Ig 1 d before immunization with 10 nmol HEL/CFA. T cell recall response at day 7 was measured by IL-2 ELISPOT shown as the percentage of total responses in distal draining lymph nodes. For statistical analysis in (E), the group of wild-type mice with control Ig was compared with other groups by Mann-Whitney t test. Shown are data from 3 (C and D) and 4 (E) individual experiments with n > 2 in each group. (A–E) Data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Pop, popliteal; iL, iliac; IN, inguinal; RE, renal; AX, axillary lymph nodes.
Figure 3.
Figure 3.
Prostanoids mediate the neutrophil effect on the magnitude and spread of T cell responses. (A and B) B10.BR mice were i.p. injected with PBS or indomethacin (INDO, 40 µg/mouse) 1 d before immunization with 10 nmol HEL/CFA. T cell recall responses were measured by IL-2 ELISPOT at day 7 after immunization. (A) The number of IL-2 spots in individual draining lymph nodes. (B) Percentage of total responses in popliteal nodes or distal draining nodes was calculated by the number of IL-2–producing cells. (C) B10.BR mice were i.p. injected with indomethacin at 3 h after 10 nmol HEL/CFA immunization. T cell recall responses in popliteal nodes or distal draining nodes were measured by IL-2 ELISPOT at day 7 after immunization. (A–C) Shown are data from three individual experiments. (D) B10.BR mice were immunized with 10 nmol HEL/CFA. The number of neutrophils in popliteal nodes was analyzed at 30 min, 3 h, or 6 h after immunization by FACS of CD11b+ Ly-6G+ cells. PBS or indomethacin was given by i.p. injections at 3 h after immunization, and the number of neutrophils was analyzed at 6 h after immunization. Each dot represents a single mouse from one representative experiment. (E) C57BL/6, COX-1−/−, or COX-2−/− mice were i.p. injected with 1A8 Ab or control Ig 1 d before 10 nmol OVA/CFA immunization. T cell recall responses in the popliteal nodes or distal draining lymph nodes were measured by IL-2 and IFN-γ ELISPOT at day 7 after immunization. Shown is the percentage of total responses in distal draining lymph nodes analyzed as described. For statistical analysis in E, the group of C57BL/6 mice with control Ig was compared with other groups. (E) Data from 4 individual experiments with n = 3 in each group. (A–E) Data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney t test.
Figure 4.
Figure 4.
Prostanoid production from neutrophils. Bone marrow neutrophils from B10.BR mice were isolated by Percoll gradient followed with anti–Ly-6G microbeads. (A) Purity of neutrophils was analyzed by FACS of CD11b+ Ly-6G+ cells or Hema 3 stain for the nuclear morphology. Bar, 50 µm. Shown is one representative experiment. (B) Neutrophils (3 × 105) were stimulated with either PMA (10 µM) for 30 min, or 10mg/ml M. tuberculosis H37Ra for 2 h at room temperature. Total RNA was isolated for qRT-PCR analysis of Ptgs-1 (left) or Ptgs-2 (right) gene expression. Gene expression was shown relative to neutrophils in PBS alone. Shown are data from 3∼5 individual experiments with n > 2 each group. (C) Neutrophils (2 × 106) were cultured in PBS with the stimulation by 100 µM of PMA for 2 h at room temperature. In the control group, neutrophils were cultured with PBS alone. Exogenous arachidonic acid (40 µM) was added into the culture as the substrates. After 2 h of incubation, the reactions were stopped by methanol and the supernatants were collected for mass spectrometry analysis. Shown are pool results from three individual experiments. (B and C) Data were shown as mean± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney t test. (D) Popliteal nodes were harvested from C57BL/6 or G-CSFR−/− mice at 2 h after immunization with 10 nmol OVA/CFA and examined for prostanoid production by mass spectrometry. Shown are fold changes relative to unimmunized C57BL/6 mice. Shown is of one single experiment with n = 3 in each group. The level of PGI2 was undetectable from the samples.
Figure 5.
Figure 5.
Prostanoids from neutrophils restricted the spread of T cell responses to distal draining lymph nodes. (A) Bone marrow neutrophils (2 × 106) were adoptively transferred into G-CSFR−/− recipients by footpad injection 20 min before immunization with 10 nmol OVA/CFA. In the group of INDO-PMN, neutrophils were preincubated with 0.2 mg/ml indomethacin for 15 min at 37°C before adoptive transfer. Popliteal nodes were harvested at 2 h after immunization. The number of transferred neutrophils was analyzed by FACS of CD11b+ Ly-6G+ cells. Each dot represents one single mouse of a representative experiment. (B) G-CSFR−/− mice were transferred with wild-type bone marrow neutrophils or PBS and immunized with 10 nmol OVA/CFA. Immunized C57BL/6 wild-type mice were used as control group. T cell recall responses in popliteal nodes were measured by IL-2 ELISPOT at day 7 after immunization. Shown is data from 2 individual experiments with n = 2 in each group. (C) Bone marrow neutrophils (2 × 106) from C57BL/6 wild-type, COX-1−/− or COX-2−/− mice; bone marrow monocytes from wild-type mice, or in vitro indomethacin-treated neutrophils were transferred into G-CSFR−/− recipients immunized with 10 nmol OVA/CFA. PBS injected wild-type or G-CSFR−/− mice were used as a control group. The percentage of total T cell recall responses in the distal draining nodes was analyzed by ELISPOT at day 7 after immunization. Shown are data from 5 individual experiments with n = 2 in each group. (B and C) Data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney t test.
Figure 6.
Figure 6.
Thromboxane in neutrophil-mediated effects. (A) B10.BR mice were i.p. injected with indomethacin (INDO, 40 µg/mouse) 1 d before immunization or picotamide (PICO, 2.5 mg/mouse) at 2 h before immunization with 10 nmol HEL/CFA. The number of neutrophils in the popliteal nodes was analyzed by FACS of CD11b+ Ly-6G+ cells. Shown is pooled data from three individual experiments. Each dot represents one single mouse. Statistical analysis was processed by Mann-Whitney t test. (B) G-CSFR−/− mice were adoptively transferred with bone marrow neutrophils pretreated with picotamide (2 mg/ml for 30 min at 37°C) before immunization with 10 nmol HEL/CFA. Included are results of wild-type mice treated with aPMN antibody. Shown is data from 2 individual experiments with n = 2 in each group. (C and D) B10.BR mice were i.p. injected with: 1A8 antibody or control Ig; indomethacin or PBS at 1 d before immunization with 10 nmol HEL/CFA. Picotamide was given at 2 h before immunization. (E and F) B10.BR mice were immunized with 10 nmol HEL/CFA in the footpad. At 3 h after immunization, mice were i.p. injected with indomethacin or picotamide. (G and H) G-CSFR−/− or wild-type mice were immunized with 10 nmol HEL/CFA and i.p. injected with U-46619 (15 µg/mouse) or PBS control. (B–H) T cell recall responses were measured by IL-2 ELISPOT analysis and presented as the number of IL-2 spots (C, E, and G) or the percentage of total responses in the distal draining lymph nodes (B, D, F, and H). (C–H) Shown are data from 3 separate experiments with n = 2 in each group. (A–H) Data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney t test.
Figure 7.
Figure 7.
Migration in the draining lymph nodes after neutrophil depletion. (A) B10.BR mice were injected with 10% Evans blue or PBS into the footpad 12 h after immunization with 10 nmol HEL/CFA. Popliteal nodes or distal draining nodes were removed after 30 min of dye injection. The whole lymph nodes were cultured in formamide overnight at 37°C, and the absorbance was measured at 620 nm. Shown is fold change relative to PBS injection. Pop, popliteal; iL, iliac; IN, inguinal; RE, renal; AX, axillary lymph nodes. Shown are data from 3 individual experiments with n = 2 in each group. (B–E) Splenic CD4+ T cells (2 × 106) from 3A9 mice were adoptively transferred into the footpad of B10.BR wild-type or G-CSFR−/− recipient mice under B10.BR genetic background. The recipient mice were either immunized with 10 nmol HEL/CFA (B, C) to follow antigen-specific T cells; or injected with CFA alone (D and E) to follow non–antigen-specific T cells. At 3 (B, D) or 22 h (C, E) after cell transfer, the number of 3A9 T cells in individual draining lymph nodes was measured by FACS analysis of CD4+ 1G12+ cells. (B–E) Shown is the percentage of total transferred 3A9 T cells in each individual lymph node. (B–E) Data are from 2 individual experiments with n = 2 in each group. (F and G) B10.BR mice were injected with control Ig or 1A8 Ab 1 d before immunization with 10 nmol HEL/CFA. Lymph nodes were harvested at indicated time points. The numbers of CD3+ cells or CD19+ cells were analyzed by FACS. Shown is the percentage of T cells (F) and B cells (G) in the individual nodes. (F and G) Data are from one experiment with n = 3 in each group. (H) 3A9 mice were i.p. injected with either 1A8 Ab or control Ig 1 d before 10 nmol HEL/CFA immunization. Total CD4+ T cells were isolated from popliteal nodes at indicated time for mRNA extraction and qRT-PCR. Gene expression of S1P1 was analyzed by fold increase normalized with CD4+ T cells from unimmunized mice. Shown are data from 2 individual experiments with n = 2 in each group. (I) T cell recall responses in immunized wild-type or IFNAR−/− mice were measured by IL-2 ELISPOT at day 7 after immunization. Shown are data from 2 individual experiments with n = 2 in each group. (J) G-CSFR−/− or wild-type B10.BR mice were i.p. injected with 50 µg/mouse FTY-720 or PBS and immunized with 10 nmol HEL/CFA. T cell recall responses were measured by IL-2 ELISPOT at day 7 after immunization. Data shown are results from 3 individual experiments with n = 2 in each group. Identical results were obtained from injecting FTY-720 every 3 d until day 7 after immunization. (I and J) Shown is the percentage of total responses in distal draining lymph nodes. (A–J) Data were presented as mean ± SD. Statistical analysis was processed by Mann-Whitney t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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