Impaired lymphatic contraction associated with immunosuppression

Abstract

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b(+)Gr-1(+) cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b(+)Gr-1(+) cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.

Fig. 1.

Popliteal lymphatic vessels exhibit consistent pumping activity. (A) DsRed-labeled SMCs (Right) exhibit typical coverage of a lymphatic vessel (LV) identified by lymphangiography (Left, green). The adjacent blood vessel (BV) has greater smooth muscle cell coverage. (B) Representative lymphatic contraction curves show reduced contraction in eNOS^−/− and l-NMMA–treated mice compared with WT and iNOS^−/− mice. (C) Lymphatic contraction analyses of diameter, frequency, amplitude, ejection fraction, and lymphatic output. , C57BL/6, n = 21; , iNOS^−/−, n = 14; , eNOS^−/−, n = 27; , l-NMMA, n = 11. *P < 0.05. P values are listed in Table S1. Error bars show SEM.

Fig. 2.

Inflammation modulates lymphatic contraction through iNOS. (A) Representative lymphatic contraction curves for WT, eNOS^−/−, and iNOS^−/− mice during inflammation. Only PLVs in iNOS^−/− mice maintain strong contractions. (B) Lymphatic contraction analyses at day 4 after OX painting. , C57BL/6, n = 14; , iNOS^−/−, n = 15; , eNOS^−/−, n = 9. (C) Immunohistochemical staining shows a large cell infiltrate of iNOS⁺ cells into the s.c. area of inflamed skin (OXd4). (D) Mice implanted with iNOS^−/− macrophages maintain strong lymphatic contractions compared with control. , WT macrophage, n = 8; , iNOS^−/− macrophage, n = 8. *P < 0.05. P values are listed in Table S1. Error bars show SEM.

Fig. 3.

Bone marrow-derived cells regulate lymphatic contraction during inflammation through iNOS. (A) BMDCs closely interact with lymphatic vessels during normal conditions (arrows). After the immunization, massive accumulation of BMDCs around lymphatic vessels occurs [green GFP⁺ BMDCs; red, rhodamine-dextran lymphangiography (Left) or αSMA⁺ cells (Right)]. (B) Representative lymphatic vessel contraction curves demonstrate that iNOS^−/− to WT BMT mice maintain strong lymphatic contraction during inflammation whereas it is impaired in WT to iNOS^−/− or WT to WT mice (Upper). On the other hand, there is no difference among the groups under normal conditions (Lower). (C and D) Lymphatic contraction analyses during (C) normal and (D) inflammatory conditions. , WT–WT normal, n = 8, OXd4, n = 7; , iNOS^−/−–WT normal, n = 8, OXd4, n = 9; , WT–iNOS^−/− normal, n = 8, OXd4, n = 8. *P < 0.05. P values are listed in Table S1. Error bars show SEM.

Fig. 4.

Gr-1 cells are the major iNOS-expressing cells that suppress lymphatic contraction during the OX-induced edema. (A) iNOS-expressing cells surround the lymphatic vessel, which is highlighted with FITC-Lectin (green, LV). iNOS is stained with FITC-labeled antibody (green, punctuate stain). Upper, F4/80; Lower, Gr-1 (red). (B) Gr-1 antibody treatment maintains stronger lymphatic contraction compared with Rat IgG control antibody treatment. , Control IgG, n = 8; , Gr-1 antibody, n = 9. *P < 0.05.

Fig. 5.

Induction of EAE is prevented temporarily after oxazolone skin painting due to reduced MOG accumulation in the draining lymph node. Dynamics of (A) lymphatic contraction and (B) Gr-1⁺ cell accumulation during OX contact sensitization are shown at times between 0 and 14 d. (C) Reduction of FITC-positive DC accumulation in LN when microspheres were injected at OXd2, but recovered if injected at OXd7. (D) EAE clinical score shows protection from autoimmune response when MOG was injected at OXd2, but not at OXd7.

Fig. 6.

Proposed mechanisms of NOS regulation of lymphatic contraction during normal and inflammatory conditions. Under normal conditions, eNOS in lymphatic endothelial cells produces temporal and spatial NO gradients that maintain robust lymphatic contractions (Inset). During inflammation, NO production from iNOS in infiltrated BMDCs overwhelms the NO gradients produced by eNOS. BMDC-derived NO causes excessive relaxation of collecting lymphatic vessels, thereby reducing the strength of lymphatic contraction (Inset).