CHAPTER FOUR

The Lymphatic Fluid

Laura Santambrogio1
Albert Einstein College of Medicine, New York, NY, United States
1
Corresponding author: e-mail address: laura.santambrogio@einstein.yu.edu

Contents
1. Introduction 112
2. Lymph Formation 112
2.1 Protein Ultrafiltration From the Blood Capillaries to the Interstitial Fluid 112
2.2 The Contribution of the Parenchymal Proteome to Lymph Composition 114
2.3 Lymph Role in Body Homeostasis 115
3. Lymph Circulation 116
4. Lymph Proteomics 117
4.1 Comparative Analysis Between Lymph and Plasma 117
4.2 Proteins From Intracellular Sources 120
4.3 Proteins From Extracellular Sources 121
4.4 The Lymph Proteome in Pathological Conditions 122
5. The Lymph Peptidome 124
6. Immunological Role of the Lymph-Carried Proteome and Peptidome 125
References 127

Abstract
This review will highlight our current understanding of the formation, circulation, and
immunological role of lymphatic fluid. The formation of the extracellular fluid depends
on the net balance between the hydrostatic and osmotic pressure gradients effective in
the capillary beds. Lymph originates from the extracellular fluid and its composition
combines the ultrafiltrated plasma proteins with the proteome generated by the met-
abolic activities of each parenchymal tissue. Several analyses have indicated how the
lymph composition reflects the organs’ physiological and pathological states. The col-
lected lymphatic fluid moves from the capillaries into progressively larger collectors
toward the draining lymph node aided by the lymphangion contractility and unidirec-
tional valves, which prevent backflow. The proteomic composition of the lymphatic
fluid is reflected in the MHC II peptidome presented by nodal antigen-presenting cells.
Taken together, the past few years have generated new interest in the formation, trans-
port, and immunological role of the lymphatic fluid.

International Review of Cell and Molecular Biology, Volume 337 # 2018 Elsevier Inc. 111
ISSN 1937-6448 All rights reserved.
https://doi.org/10.1016/bs.ircmb.2017.12.002
112 Laura Santambrogio

1. INTRODUCTION
The lymph is the biological fluid that derives from a combination of
the interstitial fluid with products of tissue metabolism and catabolism, apo-
ptotic cells, cellular debris, and circulating immune cells (Santambrogio and
Santambrogio, 2013). By carrying the tissue proteomic signature and invad-
ing pathogens to the draining lymph nodes, under both physiological and
pathological conditions, the lymph plays a pivotal role in every immunolog-
ical process, including maintenance of immunological tolerance, immunity
to pathogens, autoimmunity, inflammation, and cancer (Santambrogio and
Santambrogio, 2013).
Until a few years ago, the composition of the lymphatic fluid was virtu-
ally unknown, and it was generally accepted that the lymph and blood com-
position overlapped. This lack of knowledge was mostly due to the technical
difficulty in cannulating lymphatic vessels and the small amount of collected
fluid. Overall these technical problems prevented an in-depth analysis of the
lymph-circulating immune cells as well as any biochemical–biophysical
analysis aimed at analyzing the lymph composition. Overtime most of these
technical issues have been resolved and as such lymph biology has progres-
sively received more attention. Indeed in the last 15 years, progress has been
made toward an understanding of the mechanisms regulating lymph forma-
tion, circulation, and composition. In this review, I will summarize the cur-
rent knowledge on the mechanisms controlling the formation of the
interstitial fluid, its relationship with the lymph, the lymph circulation
and draining to the lymph nodes, and the overall progression in the analysis
of the lymph “omic” composition in physiological and pathological
conditions.

2. LYMPH FORMATION
2.1 Protein Ultrafiltration From the Blood Capillaries
to the Interstitial Fluid
The blood circulating throughout the capillary beds is not directly in contact
with the cellular layers of parenchymal tissues; as such proteins, lipids, and
other molecules are transported from the intravascular to the extravascular
space in order to deliver cellular nutrients and hydration to each cell
(Bird et al., 2009; Jafarnejad et al., 2015; Leak et al., 2004; Levick and
The Lymphatic Fluid 113

Michel, 2010; Randolph et al., 2016; Rockson, 2007). Molecule ultrafiltra-

tion is produced by the intravascular hydrostatic pressure, which drives pro-
teins and other molecules from the capillary bed into the extracellular space
(Levick and Michel, 2010; Santambrogio and Santambrogio, 2013) (Fig. 1).
The molecular movement is controlled by the glycocalyx on the luminal
surface of the endothelial cells, which functions as a sieve, filtrating proteins
according to their molecular size (Levick and Michel, 2010). Water and
small molecules easily pass through the glycocalyx and the intracellular clefts.
Proteins and bigger molecular complexes move across the epithelial barrier
through openings or pores in the glycocalyx (Levick and Michel, 2010).
According to the Starling principle, fluids exiting the arterial end of the cap-
illary bed will be absorbed back into the venular end of the capillary bed, but
this concept has been challenged. Indeed, measurements of fluid exchange in
the capillary beds have shown that in most tissues the balance between the

Lymphatic capillary Blood capillary

Lymphatic endothelial cells

Anchoring filaments Proteins, lipids, nutrients

Red blood cells (O)

Red blood cells (CO2) Apoptotic cells

Parenchymal cell Parenchymal metabolic catabolic products

Fig. 1 Dynamic of lymph formation. The formation of the extracellular fluid depends on
the net balance between the hydrostatic and osmotic pressure gradients effective in the
capillary beds. Lymph originates from the extracellular fluid and its composition com-
bines the ultrafiltrated plasma proteins with the proteome generated by the metabolic
activities of each parenchymal tissue. Circulating immune cells and apoptotic cells are
also present in the lymphatic fluid.
114 Laura Santambrogio

hydrostatic and osmotic pressure provides only a low level of fluid

reabsorption back into the capillary bed, and there is a net balance that over-
all favors interstitial fluid formation (Arkill et al., 2011, 2012; Levick and
Michel, 2010). Estimates made from measurements of thoracic duct lymph
flow indicate that 3–4 L of protein-enriched interstitial fluid are formed
everyday under physiological conditions in the human body. However,
since lymph fluid is also reabsorbed in the lymph node and returned to
the general circulation, via a Starling exchange mechanism, it appears that
an additional 3–4 L of interstitial fluid are likely formed daily (Kramer
et al., 1986). This interstitial fluid, which is present in every parenchymal
organ, is the precursor of the prenodal lymph (Adamson et al., 2004;
Arkill et al., 2011; Hu et al., 2000; Hu and Weinbaum, 1999; Kramer
et al., 1986; Levick and Michel, 2010; Squire et al., 2001) (Fig. 1).

2.2 The Contribution of the Parenchymal Proteome to Lymph

Composition
Several proteomic analyses carried out on bovine, ovine, rodent, and human
lymph sampled under physiological and pathological conditions have shown
that the lymph fluid collects the “omic signature” of the parenchymal organ
from which it drains (Clement et al., 2010, 2011, 2013; Clement and
Santambrogio, 2013; D’Alessandro et al., 2014; Dzieciatkowska et al.,
2011, 2014; Veenstra, 2007; Veenstra et al., 2005; Zurawel et al., 2011).
Although plasma albumin and serum globulins constitute the majority of
the lymph proteins, tissue-specific proteins are also highly represented in
the lymph proteome when compared to the plasma proteome. The most
striking differences between lymph and plasma proteomes are:
(i) extracellular matrix (ECM) proteins and products from their processing,
derived from tissue growth and remodeling, are more highly represented in
the lymph compared to the plasma, as well as, (ii) proteins resulting from
ongoing cellular metabolic/catabolic activities in each parenchymal organ,
and (iii) intracellular proteins released from apoptotic cells (D’Alessandro
et al., 2014; Fang et al., 2010; Goldfinch et al., 2008; Meng and
Veenstra, 2007; Mittal et al., 2009; Nguyen et al., 2010; Zurawel
et al., 2011).
On the other hand, proteins pivotal to the maintenance of the intra-
capillary osmotic pressure (albumin, and α1, α2, β globulins) and coagulation
factors are more highly represented in the plasma than the lymph proteome.
The Lymphatic Fluid 115

2.3 Lymph Role in Body Homeostasis

To avoid formation of tissue edema, the interstitial fluid needs to be
reabsorbed into the blood circulation (Jafarnejad et al., 2015; Michel,
2004; Rockson, 2007; Santambrogio and Santambrogio, 2013; Swartz
et al., 1999). Thus, an essential physiological question is why the interstitial
fluid is not directly taken up at the venule end of the capillary bead but, as
lymphatic fluid, will drain to one or more of the 600–800 lymph nodes dis-
tributed throughout the human body, before emptying into the thoracic
duct and the vena cava (Adair, 1985; Adamson et al., 2004; Armandola,
2003) (Fig. 1). Overall, there are several important reasons why the intersti-
tial fluid is not directly absorbed back into the general blood circulation but
is directed through the lymphatic system. In the blood, the fluid volume,
osmolality, protein concentration, pH, and electrolytes are very tightly con-
trolled, since even the slight modification in any of these parameters has crit-
ical consequences on the body homeostasis (Santambrogio and
Santambrogio, 2013). In contrast, the composition of the lymphatic fluid
can range widely in lipid and protein concentration, electrolytes, pH, and
overall cellular composition without harming organ homeostasis (Aukland
et al., 1984; Santambrogio and Santambrogio, 2013). Thus, any changes
in the tissue molecular biochemical and cellular composition, due to path-
ological processes, can be accommodated through the interstitial fluid that
drains into the lymphatic system. As such the lymph functions as a buffer
that can accommodate the metabolic and catabolic needs of each parenchy-
mal organ, without compromising body homeostasis (Santambrogio and
Santambrogio, 2013). Another fundamental function of the lymph is clear-
ance of tissue invading pathogens (Goldfinch et al., 2008). Indeed, by
draining through the nodes the lymphatic system ensures that tissue-
invading pathogens do not directly flow into the bloodstream but are cap-
tured by macrophages and dendritic cells (DC) present in the lymph nodes
(Junt et al., 2007; Randolph et al., 2016). Moreover, the lymph fluid carries
products of tissue remodeling, cellular secretion/processing, and cellular
debris to the nodal immune cells ensuring that the immune system is con-
stantly exposed to the tissue self-proteome for maintenance of peripheral tol-
erance (Clement et al., 2010, 2011, 2013, 2016; Clement and Santambrogio,
2013) (Fig. 1). Finally, immune cells circulating through the peripheral tissue
use the lymphatic system as a rapid and direct circulatory outlet between the
parenchymal organs and the lymph nodes (Randolph et al., 2008, 2016;
Santambrogio and Santambrogio, 2013). Overall several crucial functions
116 Laura Santambrogio

can be attributed to the lymphatic fluid: the control of bodily fluid homeo-
stasis, draining of the self- and nonself-proteome to the lymph nodes, and
enablement of immune cells trafficking (Randolph et al., 2016;
Santambrogio and Santambrogio, 2013) (Fig. 1).

3. LYMPH CIRCULATION
Networks of open-ended lymphatic capillaries, which collect the
interstitial fluid, are present in every parenchymal organ (Santambrogio
and Santambrogio, 2013). The lymphatic capillaries are formed by a single
layer of lymphatic endothelial cells (LEC) and a thin and incomplete base-
ment membrane (Tammela et al., 2005; Tammela and Alitalo, 2010). Inter-
cellular junctions, enriched in vascular endothelial (VE) cadherin and
platelet endothelial cell adhesion molecule 1 (PECAM1), connect the endo-
thelial cells. The junctions operate as one-way valves, which ease protein,
fluid, macromolecules, small molecules, and immune cell entry into the
lymphatic vessels (Baluk et al., 2007; Meens et al., 2014; Pflicke and Sixt,
2009; Platt and Randolph, 2013; Thomas et al., 2012). Additionally, the
abluminal membrane of the LEC is anchored to the ECM via anchoring
filaments, mostly composed by fibrillin. The filaments connect matrix pro-
teins to the endothelial cell cytoskeleton through the transmembrane
integrin and FAK molecule (Gerli et al., 2000). These connections allow
further opening of the intercellular junction during increased tissue edema,
to further facilitate fluid reabsorption.
The lymphatic capillaries fuse into increasingly larger vessels, known as
lymphatic collectors. The collectors are formed by a thicker and more orga-
nized basal membrane which supports the LEC (Tammela and Alitalo,
2010). Dispersed into the ECM surrounding, the lymphatic collectors are
lymphatic muscle cells and fibroblasts. The former comprise characteristics
of both smooth and striatal muscles and are fundamental to preserve the basic
tone of the vessel and to move the lymph flow toward the draining lymph
node (Gashev, 2008, 2010).
The steering of the lymph flow toward the node is also facilitated by a
series of bicuspid valves organized by layers of connective tissue overlaid
by LEC (Schmid-Schonbein, 1990; Vittet, 2014). The valves are unidirec-
tional with the lymph flow and are placed at anatomical intervals along the
collectors. The vessel area between two sets of valves is known as a
lymphangion. In close proximity to the valves, specialized muscle cells ini-
tiate muscle contraction through a Ca++ ion-mediated depolarization that
The Lymphatic Fluid 117

drives the muscle action potential. These cells act as the lymphatic collector
pacemaker, and initiate and propagate the contraction to all other muscle
cells. The contraction originates from the more peripheral lymphangion
and propagates toward those nearer to the lymph node. The valves open
and close in synchrony with vessel contraction. By coordinating the
lymphangion pumping activity with the opening and closing of the valves,
the lymph is precluded from backflow and will move unidirectionally to the
lymph node (Davis et al., 2008; Gashev, 2002, 2008, 2010; Muthuchamy
and Zawieja, 2008; Schmid-Schonbein, 1990).
Under physiological conditions, in sheep, it was originally reported that
the prenodal and postnodal lymph flow run at about 1–5 mL/h. However,
several differences have been reported according to the location of measure-
ment, animal body mass, and physiological state. For example in the mesen-
tery of fasting rats, prenodal lymph flow has been calculated to be around
15 μL/h, and the postnodal lymph flow of the mesenteric duct around
1.3 mL/h (Dixon et al., 2006).
However, during the peak of lipid absorption the flow in the mesenteric
duct can increase up to 13 mL/h (Tso et al., 1985).
In contrast, under inflammatory conditions, associated with pathogen
invasion, or sterile inflammation as observed in autoimmune diseases, an
increased lymph formation is observed, due to increased tissue edema.
The increased amount of interstitial fluid and, by default lymph fluid, is also
associated with neolymphangiogenesis (generation of newly developed lym-
phatic vessels from preexisting ones), increased immune cell trafficking,
from the periphery to the draining lymph node, and upregulation of
proinflammatory mediators, which alter lymphatic permeability and con-
tractility (Cromer et al., 2014; Rahbar et al., 2014; Swartz and
Randolph, 2014).

4. LYMPH PROTEOMICS
4.1 Comparative Analysis Between Lymph and Plasma
In the last decade, an increased number of proteomic analyses of the lym-
phatic fluid have been published. Following on from the original analysis
of ovine and bovine lymph, analysis of mouse, rat, and human lymphatic
fluid has been performed. Initially the goal of these analyses was to determine
the overall signature of the lymphatic fluid as compared to the plasma. Orig-
inally, it was thought that the protein composition of the two biological
fluids overlapped. As more proteomic analyses were performed it became
118 Laura Santambrogio

clear that was not the case and the lymph presented a molecular signature
which differed from that of the plasma. Lately, as it became apparent that
the lymphatic fluid proteome, as opposed to the plasma proteome, more
closely represented the tissue composition/proteomic from which it dra-
ined, another important goal became to use the lymphatic fluid as a liquid
biopsy during pathogenic conditions. Indeed the ultimate goal would be the
mapping of protein biomarkers and the overall molecular proteomic signa-
ture as observed in different pathologies (Clement et al., 2010, 2013, 2016;
D’Alessandro et al., 2011, 2014; Dzieciatkowska et al., 2011, 2014; Omenn
et al., 2005; Veenstra, 2007; Veenstra et al., 2005).
The original idea that the lymph was only a simple product of blood
filtration and its composition coincided with that of the plasma was mostly
due to the low sensitivity and resolution of the early mass spectrometry
instruments which could only map very abundantly expressed proteins, such
as albumin and globulins, as present in both biological fluids (Leak
et al., 2004).
In the last 5 years more accurate mapping of human, mouse, and rat
lymph, performed under physiological or pathological conditions revealed
with greater detail the complexity and distinctive characteristics of this
biological fluid. Thus far over 2000 proteins have been mapped by proteo-
mic analyses performed in different species, including humans, mice, ovine,
bovine, and swine.
Leak and colleagues performed the first proteomics analysis on ovine-
matched plasma and lymphatic fluid. By using a combinatorial approach that
included 2-DIGE with MS/MS analysis they identified a few proteins,
including glial fibrillary acidic protein and neutrophil cytosol factor 1, as pre-
sent in much higher abundance in lymph compared with plasma (Leak et al.,
2004). Their analysis was the first to report that the lymph proteome did not
completely overlap with that of the plasma, albeit the low sensitivity of the
mass spectrometer at that time did not permit for a more wide-ranging
analysis. Additionally a quantitative analysis, on rat mesenteric lymph col-
lected in fed and fasting animals, mapped close to 200 proteins involved
in innate immune responses and tissue metabolic pathways (Mittal et al.,
2008, 2009).
Our laboratory was the first to perform a proteomic analysis of the
human lymph. Our analysis established that the lymphatic fluid is enriched
in tissue-derived proteins (Clement et al., 2010, 2011, 2013, 2016; Clement
and Santambrogio, 2013; D’Alessandro et al., 2014; Dzieciatkowska et al.,
2011, 2014). We described several common proteins, present in both lymph
The Lymphatic Fluid 119

and plasma, which mostly encompassed proteins pivotal to the maintenance

of the oncotic pressure such as albumin and α1, α2, and β globulins. Other
proteins, also in common between the two fluids included the complement
system, coagulation factors, lipid transporters, and regulators of cellular
metabolism and protease inhibitors; indicative of common functionalities
between plasma and lymph. However, the analysis also emphasized the
presence of a lymph proteomic signature which included proteins associated
with apoptosis, cellular catabolism, and vesicular transport. Many proteins
also resulted from ECM tissue catabolism (collagens, laminins, and other
ECM proteins). Two main reasons explain why this category of proteins
was particularly enriched in our analysis. First and foremost the lymph sam-
ple derived from a collector draining the foot; thus, the proteome was mostly
derived from skin, muscle, and subcutaneous tissues. Second, because the
proteomic mapping was accomplished using a less sensitive mass spectrom-
eter compared to the ones available today, the results were slanted toward
more abundant molecular species (Clement and Santambrogio, 2013;
Clement et al., 2013).
Related analyses of patient-matched plasma and lymph collected from
subjects following abdominal trauma reported several tissue-specific proteins
specifically upregulated in the lymph. In matched lymph vs plasma trauma
samples over 100 proteins including products of cellular necrosis, mediators
of acute phase response, and proinflammatory responses, and immune mod-
ulators could be mapped in the lymph but not the plasma (D’Alessandro
et al., 2014; Fang et al., 2010). The lymphatic fluid was also particularly
enriched with molecules which stimulate vascular activity, as is often seen
following trauma with associated bleeding. Metabolic and catabolic products
related to energy/redox metabolism, often observed following tissue injury
were also reported. Additionally, degradation products of ECM catabolism
were also enriched in the lymph compared to the plasma (D’Alessandro
et al., 2014; Fang et al., 2010).
As methodological progress in proteomic technologies eased the map-
ping of particularly low abundance molecular species in composite biolog-
ical fluids, several of the proteins previously identified as distinctive to the
lymphatic fluid have now been reassigned in the nonredundant list of the
plasma proteome, as part of the Human Plasma Proteome Project (Ahn
et al., 2007; Ahn and Simpson, 2007; Anderson et al., 2004; Omenn
et al., 2005; Veenstra et al., 2005; Xu and Veenstra, 2008).
As the mass spectrometric technology has progressed with more sensitive
and accurate instruments, more precise quantitative and qualitative
120 Laura Santambrogio

differences will be appreciated as a signature of the two biological fluids

(Dzieciatkowska et al., 2014). Recently, a proteomic analysis that utilized
isotope-labeled amino acids has been used to quantify the nodal clearance
of a complex proteome. Additionally, more and more analyses of human
lymph collected under pathological conditions have shown the usefulness
of this biological fluid as compared to the plasma.

4.2 Proteins From Intracellular Sources

The most comprehensive proteomic analyses of the lymphatic fluid have
mostly focused on peripheral subcutaneous and mesenteric prenodal lymph,
since the former can be collected following a small cutaneous incision and
the latter during abdominal surgery (Clement et al., 2013; Magnotti et al.,
1999). Since the lymph composition is directly related to the organ that it
drains, and since only a few anatomical locations have been probed, it is safe
to assume that the lymph expression profile is yet to be fully mapped. Indeed,
a major difference between the plasma and the lymph is that the latter is
particularly enriched with intracellular proteins released from apoptotic
or, during pathological conditions, necrotic cells. A long list of proteins
of nuclear origin, such as histones, ribosomal proteins, and transcription fac-
tors have been reported (Clement et al., 2010, 2011, 2013, 2016; Clement
and Santambrogio, 2013). Several cytosol-resident proteins and proteins
derived from catabolism of organelles (mitochondria, ER, endo-lysosomes)
were also found in the lymph. Another category of proteins particularly
enriched in the lymphatic fluid is cytosolic enzymes, linked to carbohydrate,
amino acid and lipid anabolic, and catabolic pathways. Structural membrane
proteins and cytoskeletal proteins have also been mapped in the lymphatic
fluid. These proteins derive from apoptotic cells, which are normally
formed in each parenchymal organ and released into the lymphatic fluid,
or from necrotic cells as occurring during pathological conditions such as
infections, autoimmunity, cancer, and trauma (Clement et al., 2010,
2011, 2013, 2016; Clement and Santambrogio, 2013; D’Alessandro et al.,
2014; Dzieciatkowska et al., 2011, 2014; Fang et al., 2010; Goldfinch
et al., 2008; Guo et al., 2002; Meng and Veenstra, 2007; Mittal et al.,
2008, 2009).
Albeit few components of the subcellular proteome have been shown to
be present in the plasma as well the lymph (Omenn et al., 2005), the qual-
itative and quantitative amount present in the lymph during physiological or
pathological conditions underscores the role of the lymphatic fluid as the
The Lymphatic Fluid 121

major conduit for the tissue proteome (Clement et al., 2010, 2011,
2013, 2016; Clement and Santambrogio, 2013; D’Alessandro et al.,
2014; Dzieciatkowska et al., 2011, 2014; Fang et al., 2010; Goldfinch
et al., 2008; Guo et al., 2002; Meng and Veenstra, 2007; Mittal et al.,
2008, 2009).

4.3 Proteins From Extracellular Sources

Lipoproteins are among the most abundant proteins in the lymph and, albeit
radioactive tracer has shown that they derive from the plasma, their overall
lymphatic concentration, and composition differ from those found in plasma
(Randolph and Miller, 2014). ApoA-IV, A-I, A-II, C-III, E, and B were all
described in the lymph with different lymph/plasma ratios (Nanjee et al.,
2000). Whereas HDL and cholesterol were significantly higher in the inter-
stitial fluid and lymph, indicating that the lymph is the primary egress for
HDL-bound cholesterol reverse transport from the periphery to the blood-
stream and liver (Randolph and Miller, 2014).
ECM proteins are the structural support of each organ and perform a
fundamental role in preserving tissue morphological and anatomical integ-
rity, regulate tissue growth and remodeling, cellular division, and migration,
and provide a reservoir of cytokines, chemokines, and growth factors
(Badylak et al., 2009). ECM proteins, which form the scaffold of each paren-
chymal organ, undergo a constant turnover, and microanatomical reorgani-
zation to adjust to the organs physiological needs associated with cellular
division, apoptosis, migration, and immune cell patrolling. Each organ
dynamic homeostasis is enhanced during pathological conditions following
tissue injury due to trauma, acute, chronic inflammation, and cancer
(Badylak et al., 2009; Korpos et al., 2009; Lu et al., 2012; Shiomi et al.,
2010). Matrix metalloproteases (MMPs), a disintegrin and MMPs
(ADAMs), ADAMs with thrombospondin motifs are the major category
of enzymes that control the ECM protein turnover (Shiomi et al., 2010).
Additional secreted cathepsins have also been reported in the extracellular
milieu. As it was previously shown the total and active form levels of these
proteases increase in most of the pathological conditions reported above
(Clement et al., 2010, 2011, 2013, 2016; Clement and Santambrogio,
2013; D’Alessandro et al., 2014; Dzieciatkowska et al., 2011, 2014; Fang
et al., 2010; Goldfinch et al., 2008; Guo et al., 2002; Meng and Veenstra,
2007; Mittal et al., 2008, 2009). Indeed, as expected processed fragments
of ECM proteins are particularly enriched in the lymph as compared to
122 Laura Santambrogio

the plasma (Clement et al., 2010, 2013, 2016; D’Alessandro et al., 2014;
Dzieciatkowska et al., 2011, 2014). Several proteins including collagens,
laminins, fibrosin-1, aggrecan, mucins, fibronectin, and proteoglycans have
all been found in the lymph (Clement et al., 2013). Moreover, a vast range of
lymph-circulating peptides, generated by tissue-specific proteases including
different MMPs (especially MMP2, MMP8, MMP9, and MMP13) have also
been reported (Clement et al., 2010, 2013, 2016; D’Alessandro et al., 2014;
Gaggar et al., 2008; Wohlauer et al., 2012).

4.4 The Lymph Proteome in Pathological Conditions

The molecular signature of the lymph proteome not only reflects the ana-
tomical region from where the lymph drains, but also mirrors the molecular
signature of pathological conditions such as infections (Fernandez de Mera
et al., 2008; Goldfinch et al., 2008; Haenen et al., 2012; Naranjo et al., 2007;
Oveland et al., 2012; Popova et al., 2014), inflammation (Haenen et al.,
2012; Mittal et al., 2008, 2009; Oveland et al., 2012) (e.g., pancreatitis or
asthma), or traumatic events (trauma/hemorrhagic shock) (Diebel et al.,
2009, 2012; Fang et al., 2010; Zurawel et al., 2011) affecting the organs from
which the lymph collects.
One hundred and fifty-eight specific proteins were mapped in the mes-
enteric lymph collected following cecal ligation and puncture; an animal
model of septic peritonitis. None of these proteins was observed in the mes-
enteric lymph of healthy animals (Zhang et al., 2014). Among the identified
proteins some were significantly upregulated apolipoprotein E (ApoE),
annexin A1 (Anxa1), neutrophil gelatinase-associated lipocalin (NGAL),
S100a8, and S100a9. All these proteins are involved in lipid metabolism/
catabolism, and their upregulation was directly correlated with the gravity
of the septic inflammation (Zhang et al., 2014).
Lymph collected from animals infected with anthrax expressed a semi-
quantitatively different proteome as compared with lymph collected from
control animals (Popova et al., 2014). Following cutaneous exposure to
anthrax, upregulation of proteins associated with response to wounding,
inflammation and perturbations of hemostasis, innate immune response,
coagulation and fibrinolysis, regulation of body fluid levels, and vascular dis-
turbance were observed (Popova et al., 2014). Ovine lymph harvested from
sheep infected with the parasitic nematode Teladorsagia circumcincta signifi-
cantly upregulated gelsolin, α-1-β glycoprotein, and hemopexin as com-
pared to afferent lymph collected from mesenteric lymphatics of control
The Lymphatic Fluid 123

sheep (Goldfinch et al., 2008). Additionally, rat lymph, interstitial fluid, and
plasma samples, collected following an in vivo challenge with LPS, indicated
that the lymph and the interstitial fluid clearly presented a molecular signa-
ture characterized by several proinflammatory cytokines, such as TNF-α,
IL-1β, IL-6, IL-10, and ADAMST1, which were significantly lower in
the plasma (Oveland et al., 2012). Similarly, proteomic analysis of lymph
collected from rats with acute pancreatitis clearly mapped seven pancreatic
catabolic enzymes, (pancreatic amylase 2, pancreatic lipase, carboxypepti-
dase A2, chymotrypsinogen B, carboxypeptidase B1, cationic trypsinogen,
and ribonuclease 1) very early on, indicating the significance of lymphatic
fluid for liquid biopsy in pathological conditions. Alteration in the lymph
proteome after diisocyanates treatment was observed in an asthma mouse
model. Changes in the lymph proteome were more significant than the
serum and indicative that proteomic alteration could be observed before
the onset of clinical asthma (Haenen et al., 2012).
Similarly, mapping of human lymph collected from mesenteric vessels in
human, as well as rat and dog models for trauma-associated hemorrhagic
shock, pinpointed to the presence of several mediators known to be associ-
ated with multiple organ failure. Molecules associated with injury to VE cells
and neutrophil activation were present in the lymph but not in the plasma
(Magnotti et al., 1999). Similar contributions to the field indicate how the
triggering of coagulation, proinflammatory responses, neutrophil activation,
and protease/antiprotease impaired homeostasis is an early signature
observed in the lymph following posthemorrhagic shock both in humans
and animal models (D’Alessandro et al., 2014; Fang et al., 2010; Zurawel
et al., 2011). Overall the analysis revealed that after trauma-induced hemor-
rhagic shock, an increasing disproportion in the ratios between proteases to
antiproteases (SERPINs) was observed. The early reduction in the levels of
antiproteases was associated with a progressive increase in the amount and
activity of serine proteases and MMPs, altogether increasing clotting and
proinflammatory responses (D’Alessandro et al., 2014; Fang et al., 2010;
Zurawel et al., 2011). In addition, the release of damage-associated molec-
ular patterns has also been specifically reported in the lymph as compared to
the plasma in canine models of trauma and hemorrhagic shock (Diebel et al.,
2012). These observations, on the richness of tissue-specific damage-related
proteins in the lymphatic fluid, as observed in different animal models, was
also confirmed following analysis of human mesenteric lymph collected dur-
ing surgery for abdominal trauma (Clement et al., 2013; Dzieciatkowska
et al., 2011, 2014).
124 Laura Santambrogio

Thus, in conclusion all these studies reinforce the concept that the lymph
is the biological fluid that more closely mirrors the molecular signature of the
ongoing physiological and pathological conditions in parenchymal organs.
Quantitative proteomic analyses have further shown that tissue-specific anti-
gens are between 500 and 100 times more represented in the prenodal lymph
as compared to the plasma. As such, lymph analyses in different animal
models, as well as in a variety of human pathologies, strongly indicate that
the lymph is a unique biological fluid, with a proteomic composition differ-
ent from the plasma, and, with great, yet untapped potential for biomarker
discovery. The possibility to use lymph samples as liquid biopsy for a range of
human diseases should be considered for the discovery of early pathological
markers.

5. THE LYMPH PEPTIDOME

An endogenous peptidome has been characterized in many biological
fluids including plasma, saliva, urine, and the lymph, which comprises a rich
peptidome (Clement and Santambrogio, 2013; Ling et al., 2010; Sturm
et al., 2013). The majority of these peptides have been sequenced and, as
expected, they have been shown to originate from intracellular organelles
(nuclei, mitochondria ribosomes, and endosomes) as well as the endoplasmic
reticulum, Golgi apparatus, and cytosol. Furthermore, peptides and degra-
dation products originated from the processing of plasma membrane recep-
tors, cytokines, chemokines, immunomodulators, coagulation factors, and
ECM proteins have also been found in the lymph (Clement et al., 2010,
2011, 2013, 2016; Clement and Santambrogio, 2013).
Contrasting the mapped peptidome with several of the available data-
bases (MEROPS, BRENDA, and CutDB) indicated that a great variety
of processing enzymes were responsible for cleaving the lymph-carried pep-
tides (Dittwald et al., 2012; Igarashi et al., 2007; Rawlings et al., 2006).
Among those, most notable are: MMPs, cathepsins, caspases, enzymes
involved in innate immune responses such as angiotensin-converting
enzyme, complement factor I, granzymes, and enzymes of the coagulation
cascade including thrombin, plasmin, and kallikreins. Altogether the char-
acterization of the variety of these processing pathways highlights the
abundance of the metabolic/catabolic processes taking place in every tissue
such as ECM degradation, processing/cleavage of surface receptors,
The Lymphatic Fluid 125

endosomal/lysosomal processing, and cellular apoptosis (Clement et al.,

2010, 2011, 2013, 2016; Clement and Santambrogio, 2013).
Very few of the lymph-carried peptides have been quantified and their
concentration is assessed to be in the low-nanomolar to low-micromolar
range. Peptides in the low-nanomolar range mostly derive from tissue anti-
gens, whereas the ones in the low-micromolar range mostly derive from the
processing of abundant proteins such as collagens and ECM proteins
(Clement et al., 2010, 2011, 2013, 2016; Clement and Santambrogio,
2013). Importantly, it has been shown that both lymph and plasma peptides
do not circulate as free peptides but are bound to several chaperones such as
albumin, lipoproteins, and transthyretin (Geho et al., 2006). Thus, the pep-
tide concentration is dictated, at least in part, by the half-life of the carrying
protein (generally between 4 and 20 days) since the peptides are in equilib-
rium between their free and bound forms (Geho et al., 2006).

6. IMMUNOLOGICAL ROLE OF THE LYMPH-CARRIED

PROTEOME AND PEPTIDOME
The lymph-transported proteins and peptides, reflecting the proteo-
mic footprint from the organ where the lymph is collected, will encounter
several professional and nonprofessional antigen-presenting cells during
their travel to the draining lymph node. Among those are tissue-resident
DC and macrophages, LEC, lymph migratory DC, circulating monocytes,
B cells, and cells from the monocyte–macrophage–DC lineages that are pre-
sent in the lymph node. Lymph-carried proteins will be taken up through
fluid phase and receptor-mediated phagocytosis by the endolysosomal sys-
tem for processing and loading in the MHC II processing machinery.
Preprocessed peptides can be directly loaded on MHC molecules either
at the cell surface or in the early endosomes (De Bruijn et al., 1991;
Nygard et al., 1994; Ploegh, 1992; Santambrogio et al., 1999a, b;
Santambrogio and Santambrogio, 2013; Schumacher et al., 1990). Proteins
phagocytosed by the different antigen-presenting cells and transported to the
endo/lysosomal compartments will produce an MHC II peptidome mostly
through cathepsin processing (Stern et al., 2006; Stern and Santambrogio,
2016). Lymph-circulating peptides have been shown to be generated by a
variety of processing pathways, including MMPs, calpains, caspases, and
granzymes (Clement et al., 2010; Goldschneider and Cone, 2003; Pang
et al., 2009; Shen et al., 2006, 2010; Stern and Santambrogio, 2016). These
126 Laura Santambrogio

peptides can be directly loaded on MHC I and MHC II surface molecules or

in early endosomes (Eisen et al., 2012; Eisenlohr, 2013; Santambrogio et al.,
1999a, b; Schumacher et al., 1990; Villadangos et al., 2000). Thus, together
the endosomal and extracellular processing pathways will produce a diverse
array of peptides from the same protein, increasing, qualitatively and quan-
titatively, the overall presented peptidome (Stern and Santambrogio, 2016).
The self-proteome, degradome, and peptidome carried by the lymphatic
fluid can contribute to the maintenance of central and peripheral tolerance
(Bonasio et al., 2006; Campbell et al., 2009; Chowdhury et al., 1995a, b;
Donskoy and Goldschneider, 2003; Gallegos and Bevan, 2004;
Goldschneider and Cone, 2003; Idoyaga et al., 2013; Liblau et al., 1996;
Lovitch et al., 2003, 2007; Oluwole et al., 1995; Shimomura et al., 1995;
Strong and Unanue, 2011; Volkmann et al., 1997; Zal et al., 1994). It has
been shown by several groups that migratory DC ferry antigens from the
peripheral circulation to the thymus and play a major role in T cell negative
selection. Additionally, they also carry antigens into the lymph node for
maintenance of peripheral tolerance. At the same time, other reports showed
that nodal DC can process and present the incoming proteome to induce
peripheral T cell anergy and Treg differentiation (Donskoy and
Goldschneider, 2003; Goldschneider and Cone, 2003; Idoyaga et al.,
2013; Lovitch et al., 2003; Volkmann et al., 1997). Conversely, several
papers have also reported that processing of tissue-specific antigens is
involved in the development of autoimmunity (Lovitch et al., 2003;
Strong and Unanue, 2011). Several elements contribute to the tilting of
the balance from tolerance to autoimmunity such as increases in copy num-
ber of presented epitopes, changes in the type of antigen-presenting cells, de
novo generation of T cells epitopes by different processing pathways, and
changes in MHC II binding affinity/stability. To confirm this hypothesis,
several proteomic analyses have reported both qualitative and quantitative
changes in the proteome composition between physiological and patholog-
ical conditions. Quantitative changes in protein amounts could affect the
number of presented epitopes from a given protein. In addition, during
pathological conditions changes in tissue proteases have also been reported
which could facilitate neoepitope generation. Inflammatory conditions by
changing the redox environment may also contribute to protein
carbonylation as well as other posttranslational modifications, which facili-
tates neoepitope formation as well as peptide affinity for MHC class I and
MHC class II molecules.
The Lymphatic Fluid 127

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