W2A-026 Lymph system-0.txt
III. THE FORMATION OF LYMPH

The formation of lymph is influenced by the relative permeabilities of the blood capillary endothelium and the lymphatic capillary endothelium. Normal blood capillaries are impermeable to proteins of molecular weight less than 40 kD which may leak out and accumulate in the interstitium. Persistent leakage and accumulation would eventually result in edema formation. To avoid this situation, the lymphatic system acts by removing the excess tissue fluids and plasma proteins, i.e., lymph, and returning it to the bloodstream. In this way the homeostasis of the interstitial environment is maintained. The permeability of the blood capillary endothelium is limited. Although freely permeable to water and molecules less than 40 kD molecular weight, it is only semi permeable to colloid of protein molecular size. Consequently, only relatively small amounts of the total plasma protein cross the blood capillary wall.

A. PERMEABILITY OF THE LYMPHATIC CAPILLARY ENDOTHELIUM

The pathways taken, and the forces involved in the movement of interstitial fluid and proteins (lymph) across the lymphatic endothelium into the lumen of the capillaries, are the subject of ongoing research. Two major pathways have been suggested; the first is via intercellular junctions and the second via cytoplasmic vesicles. Most of the work to determine the relative contribution of the two routes to lymphatic capillary transport has been carried out using macromolecular tracers.

1. Intercellular Junctions

The role of the intercellular junction was investigated by Leak following interstitial injection of ferritin, thorium dioxide, carbon, and latex spheres. The results obtained indicate that these tracer substances are transported into and out of lymph via the intercellular junctions rather than by vesicular transport (Figure 6). These results have been supported by numerous other studies.

The relative contribution of the two routes has also been examined following intravenous (I.V.) injection of tracers such as horseradish peroxidase (HRP) and ferritin. These experiments, performed using renal lymphatics, are more physiologically relevant since the possibility of producing artificially high interstitial pressure is avoided by intravenous administration. The choice of HRP is particularly relevant since the possibility of producing artificially high interstitial pressure is avoided by intravenous administration. The choice of HRP is particularly relevant since this molecule has a molecular weight of 40 kD and a diameter of 5mm which is relatively close in size to that of plasma albumin (69 kD and a diameter of 7 nm). The results showed no significant difference in the proportions of widths of the intercellular junctions after administration of HRP or ferritin compared to the control The tracer molecules entered renal lymphatics rapidly after I.V. injection and were transported in part via normal (20 to 30 nm) intercellular spaces between adjacent endothelial cells.

Chylomicrons, which have a much larger diameter of approximately 200 nm, and are secreted in large numbers following a fatty meal, are transported into the intestinal lymphatics (lacteals) partly by the intercellular route (Figure 7).

2. Vesicles

The studies described above of intercellular transport simultaneously assessed the role of vesicular transport, and the results imply that vesicular transport is also in important route for translymphatic transport and lymph formation. Following I.V. injection of HRP, the volume density (i.e., the number) of small vesicles increased significantly and many of the vesicles contained the tracer. The distribution of vesicles within the cytoplasm did not differ significantly from that of controls, thus indicating no major change in the mechanism of vesicular formation and movement. Yang et al. therefore concluded that vesicles, if involved in translymphatic transport, may be stimulated by even a small increase in protein, in this study HRP, in the interstitium.

O'Morchoe et al. developed an in vitro model using the canine renal hilar lymphatics to study the temperature dependence of protein transport across the lymphatic endothelium. The rationale behind this study was that if vesicular transport does occur then the rate of protein movement would be temperature dependent, in keeping with the known effect of temperature on vesicles, i.e., a decrease with decreasing temperature. In contrast, if the protein moves only by diffusion between adjacent cells then temperature would have little relative effect on the rate. The results demonstrated that the rate of protein transport was influenced by temperature, being significantly reduced by 49 31% and 29% when the temperature was lowered to 4 24 and 30 C, respectively, from an initial temperature of 37 C. When the temperature was raised to 48 C, the rate was significantly increased by 48%. No significant changes in intercellular junctional or vesicular parameters were found between the control lymphatics and those perfused at 24, 30 and 40 C, thus indicating that the integrity of the tissues was maintained. At 4 C vesicular size decreased, the number of free and luminal surfaces decreased. The authors concluded that the results obtained support the existence of a vesicular lymphatic transendothelial route.

Dobbins and Rollins demonstrated the role of vesicles in chylomicron transport in the intestinal lymphatics (Figure 7). Their results showed chylomicrons present within large pinocytotic vesicles opening to the abluminal side of the endothelia, others were completely enclosed within lacteal endothelia, and some were apparently "emptying" into the lacteal lumen. They concluded that the vesicular route is quantitatively more important than the intercellular route in the translymphatic transport of macromolecules including chylomicrons.

While results have been published in support of both intercellular and vesicular routes of transport across the lymphatic endothelium, the relative contribution of each individual route is not clear. It appears, however, that both routes function simultaneously (Figure 8) and the importance of each may vary in different regions of the body and under different physiological conditions.

IV. LYMPH FLOW

Lymph flow or circulation can be broken down into two stages. The first stage involves lymph absorption and flow at the lymphatic capillaries via the intercellular or vesicular route. The second stage involves propulsion of the lymph along the larger contractile collecting lymphatic vessels.

A. LYMPHATIC CAPILLARIES

Different theories have been proposed to describe lymph filling or flow into the lymphatic capillaries. At the lymphatic capillary level there is extensive overlapping of adjacent endothelial cell walls. In addition, numerous anchoring filaments (af) are attached to the abluminal cell wall, except for the inner segments of the overlapped junction (Figure 9). This arrangement creates a flap valve (trap door) effect which is free to move in response to pressure changes. As the interstitial pressure increased above that in the lymphatic lumen, this applies tension on the anchoring filaments, which in turn pull on the attached portion of the lymphatic capillary wall allowing the unsupported portion of the cell wall (i.e., the flap) to swing open into the lumen [Figure 9 (b) and (c)]. In contrast, when the lymphatic capillary luminal pressure exceeds that of the interstitium, the flap is closed against the overlapping cell wall. Therefore, the pressure differentials between the interstitium and the lymphatic capillary lumen play a vital role in lymph formation and flow.

Casley-Smith proposed an osmotic pull hypothesis to explain lymph flow through the lymphatic capillaries. This theory suggests that compression of the capillaries results in water expulsion out of the lymphatic capillaries into the interstitium through small pores impermeable to proteins. When the compressive force is released, the newly concentrated lymph exerts an osmotic force which draws fluid back into the capillaries via large pores. Considerable doubt has been cast on this theory due to recent studies which showed that protein concentration does not vary with vessel size and that lymph protein concentration is the same as that in the tissue.

An alternative possibility, namely the suction theory, has been proposed. In this case it is argued that an increased in the interstitial pressure will in turn cause the intraluminal lymphatic pressure to rise. If the pressure in the lymphatic capillaries is greater than in the adjacent lymphangion (i.e. the segment between two adjacent valves), then lymph will flow from the capillaries into the lymphagion. Once the pressure within the larger vessel reaches its threshold, an active contraction is initiated in the lymphangion which propels the lymph forward into the adjoining lymphangion. During recovery from the contraction, the pressure within the lymphangion becomes negative and thus draws in further lymph from the capillaries. Consequently, in a type of chain reaction, the pressure within the lumen of the lymphatic capillaries becomes negative with respect to the tissue pressure, thus creating a suction within the lumen which starts the filling cycle over again.
B. COLLECTING LYMPHATIC VESSELS
In the collecting/larger lymphatic vessels, the walls become progressively thicker due to the appearance of connective tissue and smooth muscle. The thickest walls reveal a trilaminar structure similar to that of blood vessels. The intima consists of endothelium and the media contains smooth muscle cells laid down in one or two concentric layers enabling the vessels to contract and thus propel the lymph upstream. The adventitia is made up of loose connective tissue containing collagen, elastin, and fibroblasts. Small blood vessels and adrenergic nerves which service the needs of the lymphatic wall have been found within the adventitia.

All of the collecting lymphatics have a generous supply of valves, which occur at regular intervals, to prevent the backflow of lymph. However, the number and size of intravalvular segments have not been clearly established as they tend to be related to the vessel size. Large variations in the structure of these valves have been reported. Lauweryns suggested a cone- or funnel-like design. Leak, using scanning electron microscopy, found bicuspid valves along the lymphatics made up to two thin leaflets, the surfaces of which are lined with endothelial cells. In the smaller lymphatic collecting vessels the valves consist only of endothelium. In the larger vessels, however, valves have several layers and contain elastic fibers. Tricuspid valves have also, on limited occasions, been reported.

The propulsion of lymph along the collecting lymphatic vessels is governed by a number of different extrinsic and intrinsic factors. Extrinsic factors include muscle contraction, respiratory movements, and movements of the intestine. It has been shown that lymph flow increases during exercise and muscular activity. Running has been shown to increase lymph flow from the leg by approximately 150 to 300% relative to the control. Browse et al. found that the pressure in the upper and lower ends of the dog thoracic duct was significantly influenced by respiration rhythm and Lee showed that intestinal motility greatly influenced lymph flow from intestines.

Although these extrinsic factors have a role, it is now considered that the intrinsic rhythmic contractions of the valved lymphatic vessels are primarily responsible for the propulsion of the lymph toward the thoracic duct. The strength and frequency of these intrinsic contractions have been related to the rate of lymph flow.

The intrinsic smooth muscle contractions of the lymphatic vessels are regulated by three major factors: (i) the transmural vessel distension, (ii) humoral mediators, and (iii) neural mediators. The segment between two adjacent valves i.e., the lymphangion, contracts as a unit with the contraction being stimulated by radial distension. A contraction will only occur when the tension in the wall exceeds a certain threshold value, this value increasing with vessel size. Each contraction lasts for a set period of time and is followed by a period of relaxation. The contractions begin in the lymphagions adjacent to the lymphatic capillaries and spread progressively through each lymphangion towards the thoracic duct. The contractile cycle of the lymphatic smooth muscle has not been extensively examined. However, it seems that there are two separate phases: (i) the diastolic phrase, during which the intervalvular segment is filling, and (ii) the systolic phase, during which the contraction develops.

Zweifach and Prather performed a detailed study of collecting lymphatics of the mesentry of the cat and the omentum of the rabbit. The results showed that contraction begins in the smaller branches and spreads progressively to the larger vessels, this process taking 4 to 5 seconds. Between 10 and 18 contraction-relaxation cycles occurred per minute with each contraction cycle lasting on average 3 to 4 seconds. The amplitude of pulsations ranged from 2 to 4 cm H2O in the larger lymphatics and the intraluminal pressure increased gradually with vessel size.
