Murine neutrophils present Class II restricted antigen - PubMed (original) (raw)

Murine neutrophils present Class II restricted antigen

Shauna Culshaw et al. Immunol Lett. 2008.

Abstract

Neutrophils were originally described as short lived, terminally differentiated phagocytes that contribute only to the innate immune response. Recent evidence of neutrophil cytokine production and expression of numerous cell surface proteins has suggested that neutrophils are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Under certain inflammatory conditions human neutrophils express major histocompatibilty complex (MHC) Class II and the costimulatory molecules CD80 and CD86. We have employed a murine T cell hybridoma with a transgenic T cell receptor specific for ovalbumin peptide 323-339 (OVA(323-339)), and a green fluorescent reporter of T cell receptor ligation, to directly investigate neutrophil-T cell interactions. These cells provide an ideal model system, allowing precise identification of antigen specificity and a clear readout of T cell activation. Additionally, whilst murine neutrophils have previously been shown to stimulate MHC Class I-dependent CD8(+) T cell activation, CD4(+) T cells stimulation via MHC Class II-expressing neutrophils has not been investigated. We addressed this by isolating murine neutrophils, loading with OVA(323-339) and co-culturing with T cells specific for the OVA(323-339)/MHC Class II complex, and this resulted in T cell activation, as determined by expression of the green-fluorescent protein reporter. Antigen-pulsed neutrophils were also able to prime naïve OVA-specific CD4(+) T cells in a contact-dependent manner, as shown by proliferation and cytokine production. Activation of lymphocytes was not due to contaminating macrophages. These studies demonstrate that murine neutrophils present MHC Class II-restricted peptides and induce T cell proliferation, confirming findings in human neutrophils, and demonstrate a novel pro inflammatory effect of murine neutrophils.

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Figures

Fig. 1

MHC Class II expression by peritoneal exudate cells. Purified murine neutrophils were greater than 85% GR1 positive (a), and less than 4% F4/80 positive (b). GR-1 positive neutrophils showed low levels of MHC Class II expression (c); F4/80 positive macrophages showed relatively higher MHC Class II expression (d). Data shown are representative of three independent experiments.

Fig. 2

GFP expression by the DO11.10-GFP hybridoma following co-culture with APCs. Purified murine neutrophils (PMN) or macrophages (Mac) were cultured with 10 μg/ml OVA323–339 peptide and DO11.10-GFP cells for 14 h. Cells were washed and GFP expression was assessed by flow cytometry. ‘Background’ is a measure of GFP expression of DO11.10-GFP cells cultured with OVA323–339 alone; anti-CD3 was used as a positive control. Data are mean ± S.E.M. of three experiments. In each experiment, neutrophils were purified from thioglycollate-induced PEC pooled from 5 BALB/c mice. Macrophages were obtained from two similarly treated BALB/c mice. *p < 0.05 vs. background by Mann–Whitney U test.

Fig. 3

In vitro proliferation of DO11.10 lymph node cells. Single cell suspensions of lymph nodes were obtained from a DO11.10 transgenic mouse; neutrophils or macrophages were obtained as in Fig. 2. Cells were cultured for 72 h and proliferation was assessed by [3H] thymidine incorporation over the final 18 h of culture; cytokine production was assessed by ELISA of cell culture supernatants. DO11.10 lymph node cells were either cultured only with the stimuli indicated (a, c and e), or co-cultured with neutrophils or macrophages (b, d, and f). The ratio of APC to T cell in the culture, and whether the APC was pulsed with OVA323–339 prior to addition to the co-culture, is indicated on the _x_-axis. Data are mean ± S.E.M. of triplicate cultures. *p < 0.05 vs. medium by Student's _t_-test, representative of three similar experiments.

Fig. 4

Antigen-specific proliferation and cytokine production was cell contact dependent. Single cell suspensions derived from lymph nodes of DO11.10 mice, were stimulated with Con A, OVA323–339, or APCs (DCs or neutrophils), which had been pulsed with OVA323–339. In parallel cultures, the stimulus was separated from the responding T cells by a transwell membrane. The stimuli were placed in the upper compartment, and (a) proliferation and (b) IFNγ production by the OVA323–339-specific T cells in the lower chamber was assessed. T cells did not respond when cultured with APCs alone (data not shown). Similarly, culturing T cells in the upper chambers resulted in no measurable response in the lower chambers. Data are mean ± S.E.M. of triplicate cultures and representative of two similar experiments.

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Murine neutrophils present Class II restricted antigen - PubMed (original) (raw)