Author's response

Rebecca G. Wells and Neil D. Theise shared

Open questions 

1. You mention throughout your work, observations performed in humans, but also work done in different animals. Do you know how and if interstitial space continuity differs between species? If so, evolutionarily what are the advantages of this continuity?

Studies by Franklin Mall from 1906 show the continuity between the interstitial spaces of the cat liver portal tract stroma (the “space of Mall”),²  akin to our work suggesting interstitial space continuity. Beyond that, to our knowledge, there is little published data. We welcome communication from your readers regarding important literature that bears meaningfully on these questions.

In the absence of evidence to suggest differences in species, we think this is likely to be a conserved structure, at least in mammalian tissues.  Interstitial pathways for cell and molecular signalling between distant sites in the body could significantly enrich the potential to keep the body’s different regions in homeostasis, in complementary ways to those of the nervous and cardiovascular systems.

2. Following from the previous question, is there pathology known to arise from defects in IS interconnectivity?

As we noted in our first paper³ (Supplemental Figure 2), mechanical compression of the interstitial space during bowel obstruction, such as from hernias, torsion and volvulus, can lead to marked expansion of interstitial spaces and associated edema.³  Another example of site specific edema, associated with considerable and troubling symptoms including pain, immobility, and infection, is lymphedema following some cancer (surgical or radiologic) treatments. For example, breast cancer-related lymphedema of the arm is a common complication after treatment.⁴   Such edema is a static fluid overload of the interstitial spaces of the dermis and musculoskeletal fascia of the involved limb.  Whether it arises purely from lymphatic obstruction (“backing up” into the interstitial spaces drained by the lymphatics) or also through a component of direct interstitial injury (particularly from radiation) merits exploration.  Obstruction of the biliary tree by gallstones leads to edema of the extrahepatic and intrahepatic biliary tree, one of the conditions that predisposes to increased risk of bacterial superinfections (“ascending cholangitis”).

The group led by Antonio Stecco has suggested that altered HA content (both molecular forms and amount) in regions of the muscular fascia within paralyzed or immobilized limbs in patients with central nervous system injuries such as strokes, tumors, cerebral palsy, etc may be important in post-injury spasticity.^6,7  Injection of human recombinant hyaluronidase into the affected fascia relieves the spasticity.⁷  Thus, both internal and external causes of obstruction could lead to changes in the function of the interstitial spaces.

3. In your experiment analysing particle size relative to distance of inoculation site, you explore two hypotheses: one that envisions transport of particles within cells (eg. macrophages) into lymph nodes, and one that envisions transport via fluid in the interstitial space. What is the role of the lymph nodes and lymphatics in particle distribution, and how does it relate to the interstitial space (i.e. the interconnection between them). Can this not be a third form of distribution?

Fluid from interstitial spaces may move into the cardiovascular system from the small spaces (<10 microns) around capillaries within tissues.  We have not studied possible passage of small (nano-size) particles between these compartments.  However, as we demonstrate, ^1,3 there is connectivity from larger interstitial spaces of fibrous tissues such as dermis and submucosa into subjacent fascia  and also into the lymphatics (indeed, it should be remembered that lymph derives from interstitial fluid⁷).  Particles small enough to pass through the mix of proteoglycans/HA within the interstitial spaces can be seen distant from injection sites.¹  They may also be carried from the injection site within macrophages, both into the lymphatic drainage to the regional lymph nodes3  and into subjacent fascial layers.

4. You mention in your conclusion, the relevance of the interconnectivity between organs, including vessels and nerves. Can you expand further on the implications of the involvement of vessels and nerves?

The continuity of interstitial spaces of the skin and viscera with those of the fibrous covering (adventitia) of arteries and veins entering and leaving the tissues and those of the fibrous sheaths of nerves travelling from central to peripheral nervous systems (perineurium) implies that, structurally, there is continuity is not just between layers within organs or between neighbouring organs or tissues, but throughout the body, following the adventitia of the entire vascular tree and perineurium of the entire peripheral nervous system.  Functionally, this implies the possibility of cell and molecular signalling between distant sites. These could involve, for example, the microbiome (e.g. routes for gut-brain and gut-lung axes’ communication) and pathologic processes such cancer cell and infectious spread (e.g. necrotizing fasciitis).

5. You explore cell movement in pathology (ie metastasis). How does the interstitial space contribute to cell migration in homeostasis, and what is therefore, its key role? What are the key processes you would like to study, based on the findings you present in this work?

We have demonstrated the importance of macrophage trafficking within these spaces in engulfment and partial clearance of injected particulate matter.  Otherwise, we are interested in the fibroblast-like (vimentin+, CD34+) cells that line the supporting collagen bundle lattice of the spaces, which we have so far studied only superficially.  Many studies of interstitial spaces show other resident cell populations, though the spaces themselves were not well defined or understood.  For example, connective tissue layers such as the dermis and submucosae are known to have dendritic cells, comparatively low levels of lymphocytes when non-diseased, and frequent mast cells.  The mast cells are intriguing because there is a postulated associated between diseases of altered collagen (such as Ehlers-Danlos syndrome) and mast cell dysfunction(s).⁸  Do these interstitial spaces represent a “mast cell niche” and their structure/composition regulate this component of the immune system? This is one of the questions we hope to study in the future.