307
J. Physiol. (1977), 264, pp. 307-321 With 2 text-figure. Printed in Great Britain
THE INFLUENCE OF THE LYMPH NODE ON THE PROTEIN CONCENTRATION OF EFFERENT LYMPH LEAVING THE NODE
BY J. W. QUIN AND A. D. SHANNON From the Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., 2601,
Australia (Received 25 November 1975) SUMMARY
1. Experiments have been performed in sheep to determine the contribution of lymph formed within a lymph node to the total protein output in lymph leaving the node. 2. The lymphatic duct leaving the popliteal lymph node was cannulated and the protein and lymphocyte output in efferent lymph determined. The afferent lymph flow to the popliteal node was then diverted and lymph formed only within the lymph node collected from t~he efferent cannula. It appeared from the results that the popliteal lymph node forms lymph at the rate of approximately 1 ml. per hour and may contribute 30-50% of the protein output observed in efferent lymph. 3. The importance of lymph formation within the lymph node varied between nodes found in different regions of the body. This was due in part to the different protein concentrations in the afferent lymph to the different nodes. 4. A positive correlation was found between the protein and lymphocyte concentrations in efferent lymph from the popliteal lymph node in seven out of eleven sheep and in lymph formed within the popliteal lymph node in two out of three sheep. It is suggested that this relationship may be due to an increased transfer of plasma proteins through the post-capillary venules in the lymph node accompanying the continual traffic of lymphocytes across the wall of these vessels. The results indicated that the protein transfer across the post-capillary venules was not an indiscriminate transfer of plasma per se but a selective transport from the blood plasma compartment based on molecular size.
II-2
308
J. W. QUIN AND A. D. SHANNON INTRODUCTION
One of the major functions of the lymphatic system is the return of plasma proteins from the tissue space to the blood stream. Chemical and electrophoretic analysis of blood plasma and lymph have shown that all the proteins found in plasma are also found in lymph, although in lower concentrations in most instances (Yoffey & Courtice, 1970, pp. 105-106). However, the precise mechanisms by which proteins are transferred across the blood vessel wall to the tissue spaces and ultimately to the lymphatic vessels are still uncertain.
Via Pinocytotic vehicles
Via endothelial cell junctions
Accompanying the passage of re-circulating lymphocytes
Fig. 1. A diagrammatic summary of the mechanisms by which macromolecules are thought to be transported across the capillary and venular wall in vessels with a 'continuous' endothelium.
Fig. 1 summarizes the various mechanisms by which proteins are thought to be transferred from blood to the tissue space in vessels where there is a 'continuous' endothelium. Attention is drawn to the mechanism whereby a small amount of plasma protein may accompany a re-circulating cell in its passage across the endothelium. Such a mechanism of transfer of macromolecules has been shown to occur in the post-capillary venules of lymph nodes in the dog (Chapman & Bopp, 1970), mouse (Mikata & Niki, 1971; Van Deurs, R6pke & Westergaard, 1975) and rat (Nopajaroonsri, Luk & Simon, 1974), as well as in the post-capillary venules of the Peyer's patches in the mouse and rat (Schoefi, 1970, 1972). In previous
309 LYMPH DERIVED FROM LYMPH NODES studies, experimentally induced changes in the concentration and output of lymphocytes in efferent lymph of the sheep were shown to be accompanied by changes in the concentration and output of proteins in the lymph (Quin & Lascelles, 1975). The results suggested that a significant amount of plasma protein may accompany the migration of lymphocytes across the endothelium of post-capillary venules within lymph nodes and that this mechanism of protein transfer may in part explain the higher concentration of protein observed in efferent lymph of the popliteal node when compared to that in afferent lymph (Beh, Watson & Lascelles, 1974; Quin, Beh & Lascelles, 1974). In its passage from the interstitial space to the blood stream, all lymph passes through at least one lymph node and, on average, as many as eight to ten lymph nodes (Yoffey & Courtice, 1970, pp. 518-519). The passage of lymph through the lymph node may provide a site at which the composition of the lymph could be altered. Small molecules are able to diffuse into the blood stream within the lymph node (Mayerson, Patterson, McKee, Le Brie & Mayerson, 1962) while macromolecules are retained in the lymph stream (Mayerson et al. 1962; Casley-Smith, 1973). In addition, newlysynthesized antibody (Smith & Morris, 1970; Quin et al. 1974) catabolic products (Quin & Shannon, 1975) and lymph formed from venular and capillary filtrate within the lymph node may be added to the fluid passing through the node, thus materially altering the efferent output. The present study was undertaken to determine the relationship between cellular and protein concentrations in' efferent lymph under normal conditions and to determine the relative influence the lymph node itself has on the concentration of protein in efferent lymph leaving the node. MATERIALS AND METHODS
Animal. Non-pregnant Merino and Merino cross-bred ewes or castrated rams, aged between 1 and 4 years, were used in the experiments. The animals were housed indoors in individual metabolism cages and fed a mixture of lucerne hay and oats. Water was provided ad libitum. Surgical procedures. Various lymphatic ducts in forty-seven sheep were cannulated. The surgical procedures followed were those developed by Morris and his co-workers and the techniques employed are referred to in Smith, McIntosh & Morris (1970). Details of the first thirty-eight of these sheep and the lymphatic preparations established in each are summarized in Table 1. As shown in Table 1, both afferent and efferent lymph of the same lymph node were obtained from each of five sheep by cannulating one or two of the afferent lymphatic ducts to the node and the major efferent lymphatic duct from the node. In a further sixteen sheep, the efferent lymphatic from the node in a particular region and one or two of the afferent lymphatic ducts to the contralateral lymph node of the same region were cannulated. In each of seventeen sheep, either afferent or efferent lymphatic ducts of a particular region were cannulated but not both.
310
J. W. QUIN AND A. D. SHANNON
Details of the surgical procedures carried out on the nine sheep not included in Table 1 are given below in the experimental procedure section. An indwelling cannula was placed in an external jugular vein of each sheep at the time of the lymphatic cannulations. Preparation of immunoglobulins, radioactive labelling and assay. Pure ovine immunoglobulin G2 (IgG2) was isolated and labelled with radioactive 126I. The procedures followed for the isolation, labelling and assay of this protein have been described previously (Quin, Husband & Lascelles, 1975). TABLE 1. Details of the number of sheep and the type of lymphatic preparations established
Number of sheep in each group Efferent and afferent lymph from the same sheep A.-____
Popliteal lymph node Renal lymph node Prescapular lymph node Hepatic lymph node Prefemoral lymph node Total sheep in each group
Efferent or afferent lymph from each sheep
From From , the contra- Afferent only A. same lateral lymph lymph Both One node nodes sides side 1 15 2 3 1 1 2 3 5
16
2
2 7
A
Efferent only A
Both sides 2
One side 2 2 -
2
2 6
Experimental procedure. The sheep were allowed a minimum of 2-3 days to recover from the effects of anaesthesia and surgery before regular sampling of blood and lymph was undertaken. Lymph samples were collected into graduated measuring cylinders containing 0-01 ml. of a 1000 i.u. per millilitre heparin solution (Pularin, Evan's Medical). Lymph was collected in this manner at regular intervals for periods of 30-60 min and lymph flow estimated from the volume of lymph collected. Blood samples were taken from the jugular cannula in the middle of each period of lymph collection. An intravenous injection of 5-8 mg labelled IgG2 was given to each of three sheep in which an efferent lymphatic duct of the popliteal node had been cannulated. Following the equilibration of the labelled protein between blood and lymph, a correlation coefficient between the lymph: plasma radioactivity ratio (CL: Cr ratio for 125I-IgG2) and the concentration of lymphocytes in efferent lymph was determined on a consecutive series of samples taken from each sheep. To determine the amount of protein in efferent lymph derived from lymph formed within the lymph node, the following experimental procedure was employed on a total of nine sheep. The efferent lymphatic duct of the popliteal lymph node was cannulated in six sheep and lymph collected each day for a period of 5-6 days. The animals were then re-anaesthetized and the afferent lymphatic ducts, located beside the lateral saphenous vein, were ligated at the point where the vein passes beneath
LYMPH DERIVED FROM LYMPH NODES
311
the biceps femoris muscle. Two or three of the larger of these afferent lymphatic ducts were then cannulated. Partitioning surgery was carried out in an additional two sheep without the preceding period of efferent lymph drainage. In this group of eight sheep, no attempt was made to expose the lymph node thus avoiding the possibility of disturbing the vascular supply and drainage of the node. In one further sheep, the lymph node was exposed at the time of cannulation of the efferent lymphatic duct and the afferent lymphatic ducts to the node were then ligated, care being taken to avoid injury to the blood vessels in the area. In this sheep the afferent lymphatic ducts were additionally tied below the node and two of the larger afferent lymphatic ducts cannulated. To ensure that all the afferent lymph had been diverted away from the lymph node, 0-1 ml. 5 % Evans Blue (G. T. Gurr, London) was injected subcutaneously at several sites in the lower leg of all of the sheep. None of the injected dye appeared in the efferent lymph of the sheep. As an additional safeguard, the lower leg was massaged for a period of 5-10 min and the flow from the afferent and efferent cannulae observed. No change was observed in the efferent lymph flow, despite a considerable increase in the flow of afferent lymph. Efferent lymph (now derived from the lymph node only) continued to flow slowly from all nine sheep following the diversion of the afferent lymph away from the lymph node. Efferent lymph, derived from the lymph node, clotted in one of the sheep soon after surgery and in two other sheep on the third day after cannulation. However, in five of the sheep, including the animal in which the afferent ducts were tied off around the node, efferent lymph continued to flow for 3-4 weeks. Results from the seven sheep which were flowing freely between 2 and 6 days after the lymph had been partitioned are included in this paper and a minimum of two samples were averaged in each individual sheep. Protein determinations. Total protein concentrations in blood plasma and lymph samples were determined by the Biuret reaction (Gornall, Bardawill & David, 1949). A pooled plasma sample from six sheep, standardized by the microkjeldahl digestion method (Courtice, 1960) was used to draw up a standard curve. Aliquots (0- 1 ml.) of this standard sheep plasma were included in each series of determinations. The single radial immunodiffusion technique (Mancini, Carbonara & Heremans, 1965) was used to determine the concentration of albumin and each of the immunoglobulin classes in lymph and blood samples. Monospecific antisera for each of the immunoglobulin classes were prepared as described by Watson, Brandon & Lascelles (1972) and for albumin as described by Quin & Shannon (1975). Total cell counts and differential counts. Total white cells in blood and cells in lymph were counted using a Coulter counter, Model ZB1 (Coulter Electronics, England). Smears of blood and lymph were prepared, stained with Giemsa stain (G. T. Gurr, London), and differential cell counts carried out on the stained films. Statistical analysis of the results. Standard statistical methods were followed to determine standard errors of the mean, sums of squares and linear correlation values. Significant differences between means were determined by paired and unpaired t tests and the significance of correlation values was determined by a transformedvariable t test (Steel & Torrie, 1960). RESULTS
The protein concentration in efferent and afferent lymph The total protein lymph: plasma concentration ratios (CL Cp ratios) were determined for efferent and afferent lymph in four different regions
312 J. W. QUIN AND A. D. SHANNON of the sheep. The results presented in Fig. 2 show that, as the concentration of protein in afferent lymph increased, the difference in protein concentration between afferent and efferent lymph became smaller or insignificant. The total protein concentrations in afferent lymph to the popliteal and renal nodes were less than those observed in efferent lymph but there 1-0
0*9 o
S
0.70
'UV076~; C U
iv,
0
0~~~~~~~ 05
1A~~ ,0.4 0
-----
~X
i i
E 0 *0
4=.r
0
0.2~~~
02 01
0
0
. . Popliteal node
I . I . lI Renal nQde Prescapular node Hepatic node
Fig. 2. The lymph: plasma concentration ratios for total protein observed in efferent (@-@) and afferent (0-0) lymph of lymph nodes in four different regions of the body in sheep. Values presented are the means of two to six observations per sheep. The over-all mean for all sheep is shown by the dashed line.
was little difference in the total protein concentration to and from the hepatic and prescapular lymph nodes. Indeed, in two of the sheep with hepatic cannulations the protein concentrations were higher in the afferent lymph. It is unlikely that the protein differences observed could be accounted for by immunoglobulin synthesis in the lymph node since similar differences in the concentration ratios were obtained for both immunoglobulins and albumin (Table 2). The results in Table 2 also demonstrate that the CL: Cp ratios for individual proteins, observed in both efferent and afferent lymph from all regions, decreased with increasing molecular weight of the protein. The CL: Cp ratios for IgM (molecular weight 1,000,000) were usually lower than those observed for IgG, or IgG2 (molecular weight 160,000) which in turn were lower than those observed for albumin (molecular weight 69,000).
313 LYMPH DERIVED FROM LYMPH NODES Since most of the immunoglobulins in lymph to and from the popliteal lymph node are derived from the blood plasma (Quin et al. 1974), it was of interest to determine whether the rates of transport of different sized proteins into the lymph differed between efferent and afferent popliteal lymph. This was achieved by comparing the relative permeability coefficients for each of the immunoglobulins (CL : Cp ratio for each immunoglobulin divided by the CL: Cp ratio for albumin, cf. Garlick & Renkin, 1970) in both sources of lymph. The results demonstrated that there were no significant differences in the relative permeability coefficients of any of the immunoglobulins between efferent and afferent popliteal lymph. TABLE 2. The concentrations (mg/mi.) of albumin and various immunoglobulins in blood plasma and the lymph: plasma concentration ratios (CL: Cp ratios) for these proteins in four sources of efferent and afferent lymph and in lymph formed within the popliteal lymph node of the sheep. Values presented are means ± s.E. of mean. The number of sheep in each group is shown in brackets
IgGL Plasma 20*80 ± 0-83 concentration (27) Source of lymph Popliteal lymph node Afferent lymph (11) 019 + 003 Efferent lymph (11) 040+±003 Lymph derived solely from the 0*60 ± 0-06 lymph node (5) Renal lymph node Afferent lymph (3) 0-32 ± 0 05 046 ± 0*03 Efferent lymph (3) Prescapular lymph node 0*55 + 0*04 Afferent lymph (3) 0*54 + 0*05 Efferent lymph (3) Hepatic lymph node 0-71 + 0*06 Afferent lymph (3) Efferent lymph (3) 0*76 + 0.15
IgG2
IgM 2*44 ± 010 CL aC ratios
6*58 + 066
Albumin
27*20 + 077
020+ 0*02 041 + 003
019+ 001 033 + 0*02
026± 0*02 0*46 + 0*02
0-66 ± 6*06
052 ± 0-02
0*63 ± 004
031 ± 005 041 + 0O14
027 ± 003 031 ± 0.01
056 ± 010 0*62 ± 0.05
0*51 + 0*03 0*49 + 0*06
0*33 + 0*09 0*35 + 0*02
0*60 + 0*03 064 + 0*02
0*84 + 0*02 0.81 + 0-06
0*35 ± 0.01 0*38 + 0-04
0*99 ± 0*08 0-96 + 0.01
There were no significant differences in the total protein concentrations observed in lymph from different lymphatic ducts to the same popliteal node in the same sheep (four sheep, paired t test, P = 0.67), or in afferent popliteal lymph collected from opposite hind limbs in the same sheep (three sheep, paired t test, P = 0d12). On the other hand, differences were noted in one sheep in which two afferent lymphatic ducts to the prefemoral node were cannulated. The CL: Cp ratio for total protein observed in lymph
J. W. QUIN AND A. D. SHANNON from a superficial lymphatic duct of this prefemoral node was 0-48 while the ratio for lymph from a deep lymphatic duct collected simultaneously was 0-66. This would suggest that the protein differences in afferent lymph to a lymph node draining several different tissues may be larger than the protein differences between afferent and efferent lymph to such a node. 314
Anatomical study of the afferent lymphatics to the popliteal node The efferent lymphatic duct of the popliteal lymph node was cannulated in two sheep. In one of these sheep, two afferent lymphatic ducts were also cannulated in the opposite hind limb. The animals were allowed to recover from the effects of the operation and 7 days later re-anaesthetized. In the hind limb with the efferent lymphatic duct cannulated numerous subcutaneous and intramuscular injections of 0 I ml. 5 % Evan's Blue were made in the region of the extensor muscles of the tarsus and digits and into the gastrocnemius, biceps femoris, semimembranosus and semitendinosus muscles. The leg was then flexed and the injection sites massaged. No blue coloration was observed in the efferent lymph during the time of the experiment (45-60 min). In contrast, following several subcutaneous and deeper injections of 0 I ml. 5 % Light Green (G. T. Gurr, London) into the lower region of the opposite leg, green dye rapidly appeared in the afferent lymph. At the end of the experiment, the animals were killed and the popliteal lymph nodes removed and examined for Evan's Blue. In neither of the sheep was the dye observed within the lymph node. It is evident, therefore, that the popliteal lymph nodes in these sheep received their afferent lymph from the skin and tendon regions of the lower hind limb. Lymph formed within the popliteal lymph node The lymphocyte concentration, total protein concentration and lymph flow observed in efferent lymph before and after its partitioning into afferent lymph and lymph formed within the lymph node are shown in Table 3. Following the removal of the afferent lymph supply to the lymph node in the five sheep in which efferent lymph was monitored prior to the partitioning surgery, there were significant increases in the protein concentrations (P < 0.01) and lymphocyte concentrations (P < 0.05) in the efferent lymph, which was now being derived solely from the lymph node. Similar high protein and lymphocyte concentrations were observed in. the lymph derived from the popliteal lymph node of the one sheep in which the afferent ducts were tied off at the node. The flow of lymph derived solely from the lymph node in this sheep averaged 1-40 + 0-23 ml./hr. The partitioning of the efferent lymph did not result in any significant changes in
315 LYMPH DERIVED FROM LYMPH NODES the combined outputs for total protein and for lymphocytes, nor did it materially alter the total lymph flow. The concentrations of the various immunoglobulins and albumin were also determined in lymph derived from the lymph node and the results are shown in Table 2. TABLE 3. The total protein concentration, lymphocyte concentration and lymph flow in efferent lymph of the popliteal lymph node before and after partitioning into lymph derived from the node and afferent lymph. Values presented are means + S.E. of mean with the number of sheep in each group shown by the number in brackets Protein Lymphocyte concentration Lymph flow concentration (ml.fhr) (x 106/ml.) (mg/ml.) Before partitioning Efferent lymph (5) 28-64 + 3-32 3-87 ± 058 15-17 ± 3-66 After partitioning* Efferent lympht (7) 43-22 + 4*88 1-26 + 0 33 24*95 + 4*63 Afferent lymph (7) 18-89 ± 2-07 4-58 ± 0-60 0 34± 0-06 * Two sheep not monitored before partitioning. t Now derived solely from the lymph node; afferent ducts tied off below the node.
A relationship between the lymphocyte and protein concentrations in efferent lymph The correlation values for the concentration of lymphocytes and the CL: Cp ratio for total protein, or '25J-IgG2, in efferent lymph from the popliteal lymph node are presented in Table 4. Also shown in the Table are the correlation values for lymph flow and the CL: Cp ratios for total protein and 125I-IgG2. All of the correlation values were determined from observations on a series of lymph samples collected for periods of 30-60 min. In all sheep there was an apparent positive correlation between the lymphocyte concentration and CL: Cp ratio for total protein in efferent lymph but only in six cases was the relationship a significant one. The observation of a significant positive correlation between the lymphocyte concentration and CL: Cp ratio for 125J-IgG2 in two out of three sheep (Table 4) suggested that the positive correlation between lymphocyte and protein concentration in efferent lymph was not due to the addition of newly-synthesized protein derived from cells within the lymph node or from the re-circulating lymphocytes themselves. Similarly, a significant positive correlation between the lymphocyte concentration and the CL: CP ratio for total protein was observed in lymph derived solely from the lymph node in two of the three sheep studied. The possibility that the cellular and protein concentrations were positively correlated because
316 J. W. QUIN AND A. D. SHANNON they varied together as the lymph flow altered was not borne out by the observation that consecutive samples with the same lymph flow and different lymphocyte concentrations had significantly different protein concentrations. TABLE 4. The correlation values observed between the concentration of lymphocytes and the lymph: plasma concentration ratio (CL C. ratio) for total protein or 12I-IgG. in efferent and afferent lymph of the popliteal lymph node and in lymph derived solely from the popliteal lymph node. Also shown are the correlation values observed between the lymph flow and the CL C. ratios
Sheep no.
No. of observations
Correlation Correlation value value Flow and Cells and CL CP
CL C.
Efferent lymph
125I-IgG2
1 3 25
Total protein
0.96**
6 5 5 15 8 6
1
3 4 6 8 9 10 13 17 28
-0*51
0*75
0 84
0.89* 0.71** 0*46 0*64 0.69* 0.85**
11 8 10 8 13 12
0*24 0.83** 0.89*
-0*17 -0*37 - 0.89* - 0.61* 0*43 - 0.64* -0*54 -0'13 - 0.66* -0*45
0.95***
-
0.95*** 0-25
5
-0*61
Lymph derived solely from the lymph node
0.77* -0*12 -0*49
16 36 37
9 11 10
6 9 13 17 36 37
11 10
-
13 12
-0*11
0*11
0-18
11 11
0.15
-0*35 0*59
0-45
0.77**
Afferent lymph
*P
J. Physiol. (1977), 264, pp. 307-321 With 2 text-figure. Printed in Great Britain
THE INFLUENCE OF THE LYMPH NODE ON THE PROTEIN CONCENTRATION OF EFFERENT LYMPH LEAVING THE NODE
BY J. W. QUIN AND A. D. SHANNON From the Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., 2601,
Australia (Received 25 November 1975) SUMMARY
1. Experiments have been performed in sheep to determine the contribution of lymph formed within a lymph node to the total protein output in lymph leaving the node. 2. The lymphatic duct leaving the popliteal lymph node was cannulated and the protein and lymphocyte output in efferent lymph determined. The afferent lymph flow to the popliteal node was then diverted and lymph formed only within the lymph node collected from t~he efferent cannula. It appeared from the results that the popliteal lymph node forms lymph at the rate of approximately 1 ml. per hour and may contribute 30-50% of the protein output observed in efferent lymph. 3. The importance of lymph formation within the lymph node varied between nodes found in different regions of the body. This was due in part to the different protein concentrations in the afferent lymph to the different nodes. 4. A positive correlation was found between the protein and lymphocyte concentrations in efferent lymph from the popliteal lymph node in seven out of eleven sheep and in lymph formed within the popliteal lymph node in two out of three sheep. It is suggested that this relationship may be due to an increased transfer of plasma proteins through the post-capillary venules in the lymph node accompanying the continual traffic of lymphocytes across the wall of these vessels. The results indicated that the protein transfer across the post-capillary venules was not an indiscriminate transfer of plasma per se but a selective transport from the blood plasma compartment based on molecular size.
II-2
308
J. W. QUIN AND A. D. SHANNON INTRODUCTION
One of the major functions of the lymphatic system is the return of plasma proteins from the tissue space to the blood stream. Chemical and electrophoretic analysis of blood plasma and lymph have shown that all the proteins found in plasma are also found in lymph, although in lower concentrations in most instances (Yoffey & Courtice, 1970, pp. 105-106). However, the precise mechanisms by which proteins are transferred across the blood vessel wall to the tissue spaces and ultimately to the lymphatic vessels are still uncertain.
Via Pinocytotic vehicles
Via endothelial cell junctions
Accompanying the passage of re-circulating lymphocytes
Fig. 1. A diagrammatic summary of the mechanisms by which macromolecules are thought to be transported across the capillary and venular wall in vessels with a 'continuous' endothelium.
Fig. 1 summarizes the various mechanisms by which proteins are thought to be transferred from blood to the tissue space in vessels where there is a 'continuous' endothelium. Attention is drawn to the mechanism whereby a small amount of plasma protein may accompany a re-circulating cell in its passage across the endothelium. Such a mechanism of transfer of macromolecules has been shown to occur in the post-capillary venules of lymph nodes in the dog (Chapman & Bopp, 1970), mouse (Mikata & Niki, 1971; Van Deurs, R6pke & Westergaard, 1975) and rat (Nopajaroonsri, Luk & Simon, 1974), as well as in the post-capillary venules of the Peyer's patches in the mouse and rat (Schoefi, 1970, 1972). In previous
309 LYMPH DERIVED FROM LYMPH NODES studies, experimentally induced changes in the concentration and output of lymphocytes in efferent lymph of the sheep were shown to be accompanied by changes in the concentration and output of proteins in the lymph (Quin & Lascelles, 1975). The results suggested that a significant amount of plasma protein may accompany the migration of lymphocytes across the endothelium of post-capillary venules within lymph nodes and that this mechanism of protein transfer may in part explain the higher concentration of protein observed in efferent lymph of the popliteal node when compared to that in afferent lymph (Beh, Watson & Lascelles, 1974; Quin, Beh & Lascelles, 1974). In its passage from the interstitial space to the blood stream, all lymph passes through at least one lymph node and, on average, as many as eight to ten lymph nodes (Yoffey & Courtice, 1970, pp. 518-519). The passage of lymph through the lymph node may provide a site at which the composition of the lymph could be altered. Small molecules are able to diffuse into the blood stream within the lymph node (Mayerson, Patterson, McKee, Le Brie & Mayerson, 1962) while macromolecules are retained in the lymph stream (Mayerson et al. 1962; Casley-Smith, 1973). In addition, newlysynthesized antibody (Smith & Morris, 1970; Quin et al. 1974) catabolic products (Quin & Shannon, 1975) and lymph formed from venular and capillary filtrate within the lymph node may be added to the fluid passing through the node, thus materially altering the efferent output. The present study was undertaken to determine the relationship between cellular and protein concentrations in' efferent lymph under normal conditions and to determine the relative influence the lymph node itself has on the concentration of protein in efferent lymph leaving the node. MATERIALS AND METHODS
Animal. Non-pregnant Merino and Merino cross-bred ewes or castrated rams, aged between 1 and 4 years, were used in the experiments. The animals were housed indoors in individual metabolism cages and fed a mixture of lucerne hay and oats. Water was provided ad libitum. Surgical procedures. Various lymphatic ducts in forty-seven sheep were cannulated. The surgical procedures followed were those developed by Morris and his co-workers and the techniques employed are referred to in Smith, McIntosh & Morris (1970). Details of the first thirty-eight of these sheep and the lymphatic preparations established in each are summarized in Table 1. As shown in Table 1, both afferent and efferent lymph of the same lymph node were obtained from each of five sheep by cannulating one or two of the afferent lymphatic ducts to the node and the major efferent lymphatic duct from the node. In a further sixteen sheep, the efferent lymphatic from the node in a particular region and one or two of the afferent lymphatic ducts to the contralateral lymph node of the same region were cannulated. In each of seventeen sheep, either afferent or efferent lymphatic ducts of a particular region were cannulated but not both.
310
J. W. QUIN AND A. D. SHANNON
Details of the surgical procedures carried out on the nine sheep not included in Table 1 are given below in the experimental procedure section. An indwelling cannula was placed in an external jugular vein of each sheep at the time of the lymphatic cannulations. Preparation of immunoglobulins, radioactive labelling and assay. Pure ovine immunoglobulin G2 (IgG2) was isolated and labelled with radioactive 126I. The procedures followed for the isolation, labelling and assay of this protein have been described previously (Quin, Husband & Lascelles, 1975). TABLE 1. Details of the number of sheep and the type of lymphatic preparations established
Number of sheep in each group Efferent and afferent lymph from the same sheep A.-____
Popliteal lymph node Renal lymph node Prescapular lymph node Hepatic lymph node Prefemoral lymph node Total sheep in each group
Efferent or afferent lymph from each sheep
From From , the contra- Afferent only A. same lateral lymph lymph Both One node nodes sides side 1 15 2 3 1 1 2 3 5
16
2
2 7
A
Efferent only A
Both sides 2
One side 2 2 -
2
2 6
Experimental procedure. The sheep were allowed a minimum of 2-3 days to recover from the effects of anaesthesia and surgery before regular sampling of blood and lymph was undertaken. Lymph samples were collected into graduated measuring cylinders containing 0-01 ml. of a 1000 i.u. per millilitre heparin solution (Pularin, Evan's Medical). Lymph was collected in this manner at regular intervals for periods of 30-60 min and lymph flow estimated from the volume of lymph collected. Blood samples were taken from the jugular cannula in the middle of each period of lymph collection. An intravenous injection of 5-8 mg labelled IgG2 was given to each of three sheep in which an efferent lymphatic duct of the popliteal node had been cannulated. Following the equilibration of the labelled protein between blood and lymph, a correlation coefficient between the lymph: plasma radioactivity ratio (CL: Cr ratio for 125I-IgG2) and the concentration of lymphocytes in efferent lymph was determined on a consecutive series of samples taken from each sheep. To determine the amount of protein in efferent lymph derived from lymph formed within the lymph node, the following experimental procedure was employed on a total of nine sheep. The efferent lymphatic duct of the popliteal lymph node was cannulated in six sheep and lymph collected each day for a period of 5-6 days. The animals were then re-anaesthetized and the afferent lymphatic ducts, located beside the lateral saphenous vein, were ligated at the point where the vein passes beneath
LYMPH DERIVED FROM LYMPH NODES
311
the biceps femoris muscle. Two or three of the larger of these afferent lymphatic ducts were then cannulated. Partitioning surgery was carried out in an additional two sheep without the preceding period of efferent lymph drainage. In this group of eight sheep, no attempt was made to expose the lymph node thus avoiding the possibility of disturbing the vascular supply and drainage of the node. In one further sheep, the lymph node was exposed at the time of cannulation of the efferent lymphatic duct and the afferent lymphatic ducts to the node were then ligated, care being taken to avoid injury to the blood vessels in the area. In this sheep the afferent lymphatic ducts were additionally tied below the node and two of the larger afferent lymphatic ducts cannulated. To ensure that all the afferent lymph had been diverted away from the lymph node, 0-1 ml. 5 % Evans Blue (G. T. Gurr, London) was injected subcutaneously at several sites in the lower leg of all of the sheep. None of the injected dye appeared in the efferent lymph of the sheep. As an additional safeguard, the lower leg was massaged for a period of 5-10 min and the flow from the afferent and efferent cannulae observed. No change was observed in the efferent lymph flow, despite a considerable increase in the flow of afferent lymph. Efferent lymph (now derived from the lymph node only) continued to flow slowly from all nine sheep following the diversion of the afferent lymph away from the lymph node. Efferent lymph, derived from the lymph node, clotted in one of the sheep soon after surgery and in two other sheep on the third day after cannulation. However, in five of the sheep, including the animal in which the afferent ducts were tied off around the node, efferent lymph continued to flow for 3-4 weeks. Results from the seven sheep which were flowing freely between 2 and 6 days after the lymph had been partitioned are included in this paper and a minimum of two samples were averaged in each individual sheep. Protein determinations. Total protein concentrations in blood plasma and lymph samples were determined by the Biuret reaction (Gornall, Bardawill & David, 1949). A pooled plasma sample from six sheep, standardized by the microkjeldahl digestion method (Courtice, 1960) was used to draw up a standard curve. Aliquots (0- 1 ml.) of this standard sheep plasma were included in each series of determinations. The single radial immunodiffusion technique (Mancini, Carbonara & Heremans, 1965) was used to determine the concentration of albumin and each of the immunoglobulin classes in lymph and blood samples. Monospecific antisera for each of the immunoglobulin classes were prepared as described by Watson, Brandon & Lascelles (1972) and for albumin as described by Quin & Shannon (1975). Total cell counts and differential counts. Total white cells in blood and cells in lymph were counted using a Coulter counter, Model ZB1 (Coulter Electronics, England). Smears of blood and lymph were prepared, stained with Giemsa stain (G. T. Gurr, London), and differential cell counts carried out on the stained films. Statistical analysis of the results. Standard statistical methods were followed to determine standard errors of the mean, sums of squares and linear correlation values. Significant differences between means were determined by paired and unpaired t tests and the significance of correlation values was determined by a transformedvariable t test (Steel & Torrie, 1960). RESULTS
The protein concentration in efferent and afferent lymph The total protein lymph: plasma concentration ratios (CL Cp ratios) were determined for efferent and afferent lymph in four different regions
312 J. W. QUIN AND A. D. SHANNON of the sheep. The results presented in Fig. 2 show that, as the concentration of protein in afferent lymph increased, the difference in protein concentration between afferent and efferent lymph became smaller or insignificant. The total protein concentrations in afferent lymph to the popliteal and renal nodes were less than those observed in efferent lymph but there 1-0
0*9 o
S
0.70
'UV076~; C U
iv,
0
0~~~~~~~ 05
1A~~ ,0.4 0
-----
~X
i i
E 0 *0
4=.r
0
0.2~~~
02 01
0
0
. . Popliteal node
I . I . lI Renal nQde Prescapular node Hepatic node
Fig. 2. The lymph: plasma concentration ratios for total protein observed in efferent (@-@) and afferent (0-0) lymph of lymph nodes in four different regions of the body in sheep. Values presented are the means of two to six observations per sheep. The over-all mean for all sheep is shown by the dashed line.
was little difference in the total protein concentration to and from the hepatic and prescapular lymph nodes. Indeed, in two of the sheep with hepatic cannulations the protein concentrations were higher in the afferent lymph. It is unlikely that the protein differences observed could be accounted for by immunoglobulin synthesis in the lymph node since similar differences in the concentration ratios were obtained for both immunoglobulins and albumin (Table 2). The results in Table 2 also demonstrate that the CL: Cp ratios for individual proteins, observed in both efferent and afferent lymph from all regions, decreased with increasing molecular weight of the protein. The CL: Cp ratios for IgM (molecular weight 1,000,000) were usually lower than those observed for IgG, or IgG2 (molecular weight 160,000) which in turn were lower than those observed for albumin (molecular weight 69,000).
313 LYMPH DERIVED FROM LYMPH NODES Since most of the immunoglobulins in lymph to and from the popliteal lymph node are derived from the blood plasma (Quin et al. 1974), it was of interest to determine whether the rates of transport of different sized proteins into the lymph differed between efferent and afferent popliteal lymph. This was achieved by comparing the relative permeability coefficients for each of the immunoglobulins (CL : Cp ratio for each immunoglobulin divided by the CL: Cp ratio for albumin, cf. Garlick & Renkin, 1970) in both sources of lymph. The results demonstrated that there were no significant differences in the relative permeability coefficients of any of the immunoglobulins between efferent and afferent popliteal lymph. TABLE 2. The concentrations (mg/mi.) of albumin and various immunoglobulins in blood plasma and the lymph: plasma concentration ratios (CL: Cp ratios) for these proteins in four sources of efferent and afferent lymph and in lymph formed within the popliteal lymph node of the sheep. Values presented are means ± s.E. of mean. The number of sheep in each group is shown in brackets
IgGL Plasma 20*80 ± 0-83 concentration (27) Source of lymph Popliteal lymph node Afferent lymph (11) 019 + 003 Efferent lymph (11) 040+±003 Lymph derived solely from the 0*60 ± 0-06 lymph node (5) Renal lymph node Afferent lymph (3) 0-32 ± 0 05 046 ± 0*03 Efferent lymph (3) Prescapular lymph node 0*55 + 0*04 Afferent lymph (3) 0*54 + 0*05 Efferent lymph (3) Hepatic lymph node 0-71 + 0*06 Afferent lymph (3) Efferent lymph (3) 0*76 + 0.15
IgG2
IgM 2*44 ± 010 CL aC ratios
6*58 + 066
Albumin
27*20 + 077
020+ 0*02 041 + 003
019+ 001 033 + 0*02
026± 0*02 0*46 + 0*02
0-66 ± 6*06
052 ± 0-02
0*63 ± 004
031 ± 005 041 + 0O14
027 ± 003 031 ± 0.01
056 ± 010 0*62 ± 0.05
0*51 + 0*03 0*49 + 0*06
0*33 + 0*09 0*35 + 0*02
0*60 + 0*03 064 + 0*02
0*84 + 0*02 0.81 + 0-06
0*35 ± 0.01 0*38 + 0-04
0*99 ± 0*08 0-96 + 0.01
There were no significant differences in the total protein concentrations observed in lymph from different lymphatic ducts to the same popliteal node in the same sheep (four sheep, paired t test, P = 0.67), or in afferent popliteal lymph collected from opposite hind limbs in the same sheep (three sheep, paired t test, P = 0d12). On the other hand, differences were noted in one sheep in which two afferent lymphatic ducts to the prefemoral node were cannulated. The CL: Cp ratio for total protein observed in lymph
J. W. QUIN AND A. D. SHANNON from a superficial lymphatic duct of this prefemoral node was 0-48 while the ratio for lymph from a deep lymphatic duct collected simultaneously was 0-66. This would suggest that the protein differences in afferent lymph to a lymph node draining several different tissues may be larger than the protein differences between afferent and efferent lymph to such a node. 314
Anatomical study of the afferent lymphatics to the popliteal node The efferent lymphatic duct of the popliteal lymph node was cannulated in two sheep. In one of these sheep, two afferent lymphatic ducts were also cannulated in the opposite hind limb. The animals were allowed to recover from the effects of the operation and 7 days later re-anaesthetized. In the hind limb with the efferent lymphatic duct cannulated numerous subcutaneous and intramuscular injections of 0 I ml. 5 % Evan's Blue were made in the region of the extensor muscles of the tarsus and digits and into the gastrocnemius, biceps femoris, semimembranosus and semitendinosus muscles. The leg was then flexed and the injection sites massaged. No blue coloration was observed in the efferent lymph during the time of the experiment (45-60 min). In contrast, following several subcutaneous and deeper injections of 0 I ml. 5 % Light Green (G. T. Gurr, London) into the lower region of the opposite leg, green dye rapidly appeared in the afferent lymph. At the end of the experiment, the animals were killed and the popliteal lymph nodes removed and examined for Evan's Blue. In neither of the sheep was the dye observed within the lymph node. It is evident, therefore, that the popliteal lymph nodes in these sheep received their afferent lymph from the skin and tendon regions of the lower hind limb. Lymph formed within the popliteal lymph node The lymphocyte concentration, total protein concentration and lymph flow observed in efferent lymph before and after its partitioning into afferent lymph and lymph formed within the lymph node are shown in Table 3. Following the removal of the afferent lymph supply to the lymph node in the five sheep in which efferent lymph was monitored prior to the partitioning surgery, there were significant increases in the protein concentrations (P < 0.01) and lymphocyte concentrations (P < 0.05) in the efferent lymph, which was now being derived solely from the lymph node. Similar high protein and lymphocyte concentrations were observed in. the lymph derived from the popliteal lymph node of the one sheep in which the afferent ducts were tied off at the node. The flow of lymph derived solely from the lymph node in this sheep averaged 1-40 + 0-23 ml./hr. The partitioning of the efferent lymph did not result in any significant changes in
315 LYMPH DERIVED FROM LYMPH NODES the combined outputs for total protein and for lymphocytes, nor did it materially alter the total lymph flow. The concentrations of the various immunoglobulins and albumin were also determined in lymph derived from the lymph node and the results are shown in Table 2. TABLE 3. The total protein concentration, lymphocyte concentration and lymph flow in efferent lymph of the popliteal lymph node before and after partitioning into lymph derived from the node and afferent lymph. Values presented are means + S.E. of mean with the number of sheep in each group shown by the number in brackets Protein Lymphocyte concentration Lymph flow concentration (ml.fhr) (x 106/ml.) (mg/ml.) Before partitioning Efferent lymph (5) 28-64 + 3-32 3-87 ± 058 15-17 ± 3-66 After partitioning* Efferent lympht (7) 43-22 + 4*88 1-26 + 0 33 24*95 + 4*63 Afferent lymph (7) 18-89 ± 2-07 4-58 ± 0-60 0 34± 0-06 * Two sheep not monitored before partitioning. t Now derived solely from the lymph node; afferent ducts tied off below the node.
A relationship between the lymphocyte and protein concentrations in efferent lymph The correlation values for the concentration of lymphocytes and the CL: Cp ratio for total protein, or '25J-IgG2, in efferent lymph from the popliteal lymph node are presented in Table 4. Also shown in the Table are the correlation values for lymph flow and the CL: Cp ratios for total protein and 125I-IgG2. All of the correlation values were determined from observations on a series of lymph samples collected for periods of 30-60 min. In all sheep there was an apparent positive correlation between the lymphocyte concentration and CL: Cp ratio for total protein in efferent lymph but only in six cases was the relationship a significant one. The observation of a significant positive correlation between the lymphocyte concentration and CL: Cp ratio for 125J-IgG2 in two out of three sheep (Table 4) suggested that the positive correlation between lymphocyte and protein concentration in efferent lymph was not due to the addition of newly-synthesized protein derived from cells within the lymph node or from the re-circulating lymphocytes themselves. Similarly, a significant positive correlation between the lymphocyte concentration and the CL: CP ratio for total protein was observed in lymph derived solely from the lymph node in two of the three sheep studied. The possibility that the cellular and protein concentrations were positively correlated because
316 J. W. QUIN AND A. D. SHANNON they varied together as the lymph flow altered was not borne out by the observation that consecutive samples with the same lymph flow and different lymphocyte concentrations had significantly different protein concentrations. TABLE 4. The correlation values observed between the concentration of lymphocytes and the lymph: plasma concentration ratio (CL C. ratio) for total protein or 12I-IgG. in efferent and afferent lymph of the popliteal lymph node and in lymph derived solely from the popliteal lymph node. Also shown are the correlation values observed between the lymph flow and the CL C. ratios
Sheep no.
No. of observations
Correlation Correlation value value Flow and Cells and CL CP
CL C.
Efferent lymph
125I-IgG2
1 3 25
Total protein
0.96**
6 5 5 15 8 6
1
3 4 6 8 9 10 13 17 28
-0*51
0*75
0 84
0.89* 0.71** 0*46 0*64 0.69* 0.85**
11 8 10 8 13 12
0*24 0.83** 0.89*
-0*17 -0*37 - 0.89* - 0.61* 0*43 - 0.64* -0*54 -0'13 - 0.66* -0*45
0.95***
-
0.95*** 0-25
5
-0*61
Lymph derived solely from the lymph node
0.77* -0*12 -0*49
16 36 37
9 11 10
6 9 13 17 36 37
11 10
-
13 12
-0*11
0*11
0-18
11 11
0.15
-0*35 0*59
0-45
0.77**
Afferent lymph
*P
