Nitric oxide (NO) side of lymphatic flow and immune surveillance

The loss of cancer or bacteria detection by the immune system is traditionally looked at from the point of cell–cell or humoral mediator–cell interactions. However, immune surveillance in lymphatics critically requires cell transport along the lymphatics for entry of antigen/dendritic cells into the draining lymph node (1). Reductions in collecting lymphatic function affect all subsequent immune response. The work by Liao et al. (2), from a team well-versed in the complexities and limitations of transport in tumors, brings to light that lymphatic transport can be limited by a mechanism that is controlled by nitric oxide (NO) derived from bone marrow myeloid cells.

Initial and Contractile Lymphatics

The study is focused on the smooth muscle-equipped contractile (also denoted collecting) lymphatics in a part of the lymphatics located proximal to the initial lymphatics (2). Initial lymphatics, rich in numbers and deeply embedded in tissue parenchyma, consist of pure endothelial channels without perivascular cells (e.g., pericytes) and smooth muscle cells. They have overlapping cell junctions forming primary valves in addition to traditional secondary lymph valves, and they rely on surrounding tissue motions to achieve periodic lymph channel expansion and compression for collection of interstitial fluid and fluid transport inside the lymphatics (3).

By contrast, the contractile lymphatics are equipped with rows of bileaflet valves and contract by a specialized smooth muscle phenotype unique to the lymphatics to carry fluid from the initial lymphatics to the lymph nodes (4). Each pair of upstream and downstream valves in contractile lymphatics forms a lymphangion, facilitating unidirectional lymph fluid during periodic contraction. Contractile lymphatics have many of the vascular control mechanisms present in the arterioles, from classical myogenic contraction to neurogenic, purinergic, and endothelial-dependent and -independent controls (5–8).

An Obligatory Role of NO in Lymph Contraction

Lymphatics are grand avenues of the immune system. Dendritic antigen-carrying cells or metastatic cells coming from the initial lymphatics need to be carried by fluid flow inside the contractile lymphatics. If flow stops, the immune surveillance that relies on the delivery of antigen/dendritic cells into the lymph nodes also stops. Thus, we need to ask what mechanisms abolish the smooth muscle shortening in contractile lymphatics. Besides genetic origins or aging, an immediate case that comes to mind is inflammation. Inflammation abolishes interstitial fluid uptake by failure of primary valves in initial lymphatics (9) and independently stops smooth muscle contraction in contractile lymphatics. Using ejection fraction and lymphatic output determined from the frequency and magnitude of individual contractions (10), the work by Liao et al.

Lymphatic transport can be limited by a mechanism that is controlled by nitric oxide (NO) derived from bone marrow myeloid cells.

(2) confirms that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions but is not able to maintain the contractile activity in the inflammatory state. In inflammation, NO derived from inducible nitric oxide synthase (iNOS) expressing CD11b-positive myeloid cells attenuates lymphatic contraction. This inhibition of lymphatic contraction is associated with reduced autoreactive responses to antigen.

Contractile Lymphatics Flooded with NO

Tracing the source of NO, the work by Liao et al. (2) reports that bone marrow-derived CD11b-positive myeloid cells expressing iNOS infiltrate the tissue surrounding the contractile lymphatics after inflammation and inhibit autonomous lymphatic contraction during inflammation. After inflammation, a single purposeful source of NO from endothelium is awash from a new source of myeloid cells. Control experiments confirm this scenario: chimeric wild-type mice with iNOS^−/− bone marrow transplants are able to continue robust contractions, or NO from activated macrophages implanted over lymphatics suppresses the contractions. Removing NO produced by iNOS in bone marrow-derived dendritic cells increases contraction strength and lymph flow rates.

It is apparent from these findings that the microenvironment surrounding collecting lymphatic vessels is a determinant of lymphatic function. Under physiological conditions, NO produced by eNOS in endothelial cells is required for periodic contraction and lymph flow; removing NO caused a reduction in contraction strength. Under inflammatory conditions, iNOS overproduces NO, overwhelms the subtle flow-dependent NO production from eNOS, and prevents contraction. The situation facilitates lymph edema, reduces antigen delivery into lymph nodes, and consequently, reduces antigen-presenting cells and T-cell activation. The exact temporal and spatial cell sources of NO, in addition to total amount, are critical to its ultimate impact on lymphatic transport. iNOS can be produced continuously, whereas eNOS may be dynamically modulated by fluid shear stress on the lymphatic endothelium and therefore, assume a cyclic mode.

The work by Liao et al. (2) reports reduced lymphatic contraction strength and increased lymphatic diameter in eNOS^−/− mice and after NOS inhibition in wild-type mice. Because NO is a vessel relaxation factor, one expects a constricted lymphatic vessel after blockade. Surprisingly, Liao et al. (2) report larger lymphatic diameters in NOS-inhibited animals compared with control animals, which is different from a temporary inhibition. The molecular mechanism remains to be determined. Besides NO, cytokines and growth factor are also likely to affect collecting lymphatic contraction, and there may be other gases, like carbon monoxide, that regulate smooth muscle (11).

Cessation of Lymph Flow and Immune Evasion

These results have several implications for immune regulation in pathological conditions. Suppression of lymphatic function by CD11b-positive myeloid cells is a mechanism of self-protection from autoreactive responses during on-going inflammation. To initiate an immune response, antigen and antigen-presenting cells arrive in lymph nodes within hours on antigen encounter. Myeloid cells may accumulate at an inflammatory site and inhibit collecting lymphatic function, suppressing additional immune response to self-antigen by reducing antigen transport into the lymph node.

The disruption of lymphatic function by NO may provide a mechanism for immune evasion by infections or cancer. Mediators released from malignant tumor cells cause a significant reduction of lymphatic pump activity through generation of endogenous NO and activation of potassium membrane channels (6). Metastatic cell spread is facilitated by contractile lymphatics. Physiological control of lymph pumping and immunological control are closely linked, and disruption of lymphatic function may provide another mechanism of immune evasion by cancer or bacteria and may decrease the rate of rejection of transplanted organs. The molecular mechanisms depend on this delicate dance of specific cells around lymphatic structures and need to be studied in in vivo models.