Novel role of regulatory T cells in limiting early neutrophil responses in skin

Abstract

It is clear that CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells inhibit chronic inflammatory responses as well as adaptive immune responses. Among the CD4(+) T-cell population in the skin, at least one-fifth express Foxp3. As the skin is constantly exposed to antigenic challenge and is a common site of vaccination, understanding the role of these skin-resident Treg cells is important. Although the suppressive effect of Treg cells on T cells is well documented, less is known about the types of innate immune cells influenced by Treg cells and whether the Treg cells suppress acute innate immune responses in vivo. To address this we used a mouse melanoma cell line expressing Fas ligand (B16FasL), which induces an inflammatory response following subcutaneous injection of mice. We demonstrate that Treg cells limit this response by inhibiting neutrophil accumulation and survival within hours of tumour cell inoculation. This effect, which was associated with decreased expression of the neutrophil chemoattractants CXCL1 and CXCL2, promoted survival of the inoculated tumour cells. Overall, these data imply that Treg cells in the skin are rapidly mobilized and that this activity serves to limit the amplification of inflammatory responses at this site.

Figure 1

Histological examination of the site of B16Fas ligand cell inoculation in regulatory T cell-depleted and control mice. Mice treated either with isotype control monoclonal antibodies (GL113; a, b) or CD25-specific monoclonal antibodies (PC61; c, d) were injected subcutaneously 1 day later with 10⁵ B16FasL. At 24 hr post-injection mice were killed and the skin surrounding the injection site was collected for histology. Haematoxylin & eosin-stained 5-μm paraffin-mounted sections were generated throughout the skin. Sections (a) to (d) are representative mid-sections of cellular masses from at least four mice per group.

Figure 2

Distinct patterns in cell mass are observed at the site of B16Fas ligand cell inoculation in reulatory T cell-depleted versus control mice. Haematoxylin & eosin-stained sections were generated as described for Fig. 1 at 24 hr post-injection. Sections shown are representative of confined (a, b) and non-confined (c, d) cellular masses. The blue lines enclose the area of skin which histologically deviates from normal or PBS-injected skin. (e) The percentage of mice displaying the confined phenotype of cellular mass is given for each group. Statistical significance was evaluated by Fisher's exact test (***P < 0·001).

Figure 3

Live tumour cells observed in B16Fas ligand-injected mice. Haematoxylin & eosin-stained sections were generated as described for Fig. 1 at 4, 24 and 96 hr post-injection. (a) The total volume of cellular mass for each mouse was estimated based on the area of cellular mass on each section and the distance between them, as described in the Materials and methods. Data are shown ± SEM of at least four mice per group. (b) An example of live tumour cells in an established tumour. (c) Live cells within cellular masses of GL113-treated mice are identified as being histologically similar to those found within established tumours and are indicated by arrows.

Figure 4

Foxp3⁺ cells in skin of B16Fas ligand-injected mice. Serial sections of normal and B16FasL-inoculated skin from GL113-treated mice were stained with either haematoxylin & eosin or Foxp3-specific monoclonal antibodies. The Foxp3⁺ cells are indicated by the arrows. No stained cells were observed in the PC61-treated mice (not shown).

Figure 5

Depletion of CD25⁺ cells promotes neutrophil accumulation at the site of tumour inoculation. Mice were treated with either isotype control monoclonal antibodies (GL113; a) or CD25-specific monoclonal antibodies (PC61; b) and injected 1 day later with 10⁵ B16Fas ligand. At 24 hr post-injection, mice were killed and the skin surrounding the injection site was collected for histology. Interleukin-8 receptor (IL-8R) -stained 5-μm paraffin-mounted sections were generated to identify neutrophils. Areas of neutrophil infiltration are enclosed by the blue lines. Sections are representative of at least five mice per group at 24 hr. (c) Examples of neutrophils (indicated by arrows) which stain dark brown with multi-lobed nuclei. (d) The numbers of neutrophils, assessed as polymorphonuclear cells with IL-8R expression, are given where each symbol represents the score for one mouse. Statistical significance was evaluated by Mann–Whitney U-test (*P < 0·05). (e) The relative expression of CXCL1 and CXCL2, assessed by real-time PCR, in the skin of GL113-treated and PC61-treated B16FasL-inoculated mice.

Figure 6

Increased number of nuclear segments in neutrophils recovered from mice depleted of CD25⁺ cells. Mice were treated with either isotype control monoclonal antibodies (GL113) or CD25-specific monoclonal antibodies (s (PC61) and injected intraperitoneally 1 day later with 2 × 10⁶ B16Fas ligand. Eighteen hours later, mice were killed and the peritoneal cavity was lavaged. Cytospins generated of lavaged cells were stained and the number of segments in 100 neutrophils was assessed. Representative photographs of cytospins generated from GL113-treated (a) and PC61-treated (b) mice. (c) Examples of segmented neutrophils where the number of segments in each neutrophil is given. (d) The proportion of neutrophils with the indicated number of segments is shown. Mice were analysed individually and data shown are the mean ± SEM of four mice per group. Statistical significance between groups sharing the same letter was evaluated by Mann–Whitney U-test (*P> 0·05). (e) The average number or segments per neutrophil is given for each mouse. Statistical significance was evaluated by Mann–Whitney U-test (*P = 0·0286).

Figure 7

Neutrophils play a significant role in tumour rejection and in induction of adaptive immunity in regulatory T cell-depleted mice. (a) Neutrophils isolated from the bone marrow of mice were used directly as effectors against ⁵¹Cr-labelled B16 or B16Fas ligand cells at a ratio of 40 : 1. Bars indicate mean ± SD of duplicates and are representative of two independent experiments. (b) Mice were treated with either isotype control monoclonal antibodies (mAbs) (GL113) or CD25-specific mAbs (PC61), with or without Gr1-specific mAbs (RB6-8C5) for the depletion of neutrophils. Bars indicate the mean percentage rejection ± SEM in three and two experiments respectively, with the number of tumour-free mice/total number indicated. Statistical significance was evaluated by Fisher's exact test (**P < 0·01).