The Adipose Tissue Around Lymph Nodes

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Reptiles and amphibians have just a few adipose depots, mostly in the abdomen or around the tail. This arrangement is clearly practical for tissue whose sole function is storage because the adipose tissue can undergo large changes in mass without affecting the adjacent organs. In contrast, mammalian adipose tissue is always split into a few large and numerous small depots scattered over much of the body. In many of the minor depots, including 'yellow' bone marrow, the omentum and many intermuscular and perivascular sites, adipocytes are intimately associated with lymphoid tissue (13). Thorough studies of wild animals (9,12,14) show that the major depots, such as the perirenal and the posterior superficial depots, undergo large changes in mass, like adipose depots in lower vertebrates, while many of those associated with lymphoid tissue, such as the politeal, do not alter much even in massive obesity or emaciation. The popliteal has also been extensively studied in humans, because part of it is clearly visible over the gastrocnemius muscle of the lower leg. Its mass changes only slightly, in spite of large changes in body composition, so people with bulging thighs may have slim calves (15). This peculiar and almost universal feature of mammals remains to be explained convincingly.

Most mammalian adipose depots contain one or more lymph nodes, though the exact number varies between conspecific individuals, posing further obstacles to quantitative study. Some adipose depots, such as the mesentery and omentum, have dozens of lymph nodes embedded in them, but others, including the popliteal depot, contain only one or a few, and they may be concentrated into one corner. The microscopic structure of the adipose tissue sur rounding lymph nodes has not been investigated in detail since the work of Suzuki (16): standard his-tological techniques revealed no site-specific differences other than adipocyte size, and by the time immunocytochemical methods became available, interest in the microscopic anatomy of adipose tissue had waned. Many such depots are small, itself a disincentive to study, both because those of laboratory rodents offer very little tissue for experimental study, and because their reduction in humans would have little impact on obesity.

Lymph nodes as major sites of proliferation and dissemination of lymphocytes are a special feature of mammals: a few similar structures are found in certain birds but they are absent from lower vertebrates. They almost always occur embedded in adipose tissue, although most anatomical illustrations and models tend to conceal rather than emphasize the fact. Immunologists habitually begin all his-tological and physiological studies by 'cleaning' the adipose tissue off the node (17,18). The fact that lymph nodes and ducts are embedded in adipose tissue is disregarded in biochemical studies of lymph flow (19). The lymph ducts run through the adipose tissue and divide into numerous fine branches as they approach the node, thereby generating points of entry over much of its surface, and coming into contact with a large proportion of the adipocytes that immediately surround it. The adipose tissue associated with some nodes represents such a tiny fraction of the total that it is difficult to suppose that it could make a significant contribution to whole-body lipid supply. So why is it present at all?

The need to swell when fighting infection was, until recently presented as the main, if not the sole, reason for the anatomical association between adipose tissue and lymph nodes (17). However, adipocytes embedded in their network of collagen are not very compressible. It is difficult to see why adipose tissue should be preferred as a container for expandable nodes over a mainly extracellular, genuinely extensible material such as connective tissue (12). The lymphoid tissue of birds and lower vertebrates also expands when activated, but it is not closely associated with adipose tissue. In many species it could not be, because adipose tissue is confined to a few centrally located fat bodies, instead of, as in mammals, being partitioned into numerous small depots, where it can be associated with lymph nodes.

Since 1994, we have been exploring an alternative hypothesis: major lymph nodes occur in association with adipose tissue because the latter is specialized to serve as a regulatory and 'nurse' tissue. A simple experiment enables the lymphoid cells themselves to point out which kinds of adipose tissue they interact with most strongly (20). A standard mixture of lymphoid cells from the large cervical lymph nodes was incubated with or without a mitogen for several days with explants of adipose tissue taken from near to and away from nodes of various depots of the same animal. The number of new lymphocytes formed was estimated from incorporation of labelled thymidine, and lipolysis by the glycerol concentration in the incubation medium. Mature guinea-pigs of a large strain were used for this investigation: there is simply not enough adipose tissue in the node-containing depots of rats or mice to supply well-controlled experiments.

The presence of adipose tissue always curtails both spontaneous and mitogen-stimulated proliferation of lymphocytes, but the extent of inhibition depends greatly upon the source of the sample. In all the eight depots studied that contain one or more lymph nodes, but especially the mesentery, omentum, forearm, popliteal and cervical depots, the samples taken from near to a lymph node suppressed the formation of new lymphocytes more strongly than those taken from elsewhere in the same depot. The least effective samples were those from the perirenal, which in guinea-pigs (and most other mammals) do not contain any lymph nodes.

The same experiments revealed that lymphoid cells consistently induce more lipolysis in adipose tissue from near to nodes than in samples from elsewhere in the same depot, especially in the small intermuscular popliteal and cervical depots, and the omentum and mesentery (Figure 13.1). Co-incubation with lymphoid cells causes lipolysis to rise by more than threefold in perinodal samples, a greater increase than is observed when isolated adipocytes are stimulated with large doses of noradrenaline. Such effects are highly localized: adipose tissue from 1-2 mm around major lymph nodes may respond twice as much as neighbouring samples from just a centimetre away. Lipolysis from the perirenal is higher than all the other samples when they are incubated alone, but the presence of lymphoid cells stimulates a rise of less than 5%, a negligible increase compared to that observed in explants from the node-containing depots.

The gross anatomy of these nodes and their surrounding adipose tissue suggests an explanation for the strong local interactions. The mesenteric nodes, being the first to come into contact with material absorbed through the gut, are in the front line of defence against pathogens invading through the intestine. The omentum also contains a great deal of lymphoid tissue and is believed to remove debris from the abdominal cavity. The popliteal lymph node is the most distal of the lower limb nodes, and lymphoid cells arising from it protect the whole of the hindlimb below the knee. The cubital lymph node (in the 'forearm' adipose depot) is also located as 'the end of the line', and performs similar functions for the distal part of the forelimb.

Hands and feet (and paws and hooves) are continually exposed to abrasion and assaults from parasites and pathogens, so the nodes that serve them are nearer 'the front line' in dealing with local, minor injuries, infections and inflammations than the more centrally located inguinal and axillary ('behind arm') nodes. The popliteal depots are small, representing less than 5% of the total adipose mass in guinea-pigs and most other mammals (12), but they contain relatively large nodes. The popliteal 'space' contains a little adipose tissue around the node in all eutherian mammals, even in very lean wild animals in which nodeless depots are depleted completely, and in seals in which most of the adipose tissue is specialized as superficial blubber. Enclosing these important lymph nodes may be their main role: they do not enlarge with fattening as much as the large superficial and intra-abdomi-nal depots, and seem to be conserved in starvation (9,10,14,15).

Perirenal adipocytes respond satisfactorily to all other known local and blood-borne stimulants of lipolysis, and indeed this depot is often taken as a representative of the adipose mass as a whole, but as Figure 13.1 shows, it is atypical as far as interactions with the lymphocytes and macrophages are concerned. In guinea-pigs and many other mammals, the perirenal is among the largest of all depots and undergoes extensive changes in size as total fatness changes. Its lack of interaction with lymphoid cells may arise from the fact that it normally contains no lymph nodes, so would be unable to participate in local interactions with lymphoid cells, or may simply be a necessary corollary of its role as an energy store for the body as a whole.

The other, smaller depots expand and shrink less

Energy Store Body

Figure 13.1 Site-specific differences in spontaneous and lymphoid cell-stimulated glycerol release (20). Means + SE of glycerol in the medium after incubation with the mitogen, lipopolysaccharide for 48 h and an explant of adipose tissue. Explants were taken from far from (light bars) or near to (dark bars) lymph node(s) (or, in the case of perirenal, a knot of blood vessels) of four superficial (left group of bars), three intra-abdominal (centre) and two intermuscular (right) adipose depots with (shaded bars) or without (striped bars) lymphoid cells. All values are means of data from 10 mature adult guinea-pigs

Figure 13.1 Site-specific differences in spontaneous and lymphoid cell-stimulated glycerol release (20). Means + SE of glycerol in the medium after incubation with the mitogen, lipopolysaccharide for 48 h and an explant of adipose tissue. Explants were taken from far from (light bars) or near to (dark bars) lymph node(s) (or, in the case of perirenal, a knot of blood vessels) of four superficial (left group of bars), three intra-abdominal (centre) and two intermuscular (right) adipose depots with (shaded bars) or without (striped bars) lymphoid cells. All values are means of data from 10 mature adult guinea-pigs readily because part of their adipose tissue is conserved for special, local functions. Adipocytes prepared from the small quantity of adipose tissue surrounding lymph nodes are insensitive to fasting: as Figure 13.2 shows, spontaneous lipolysis in such adipocytes excised from guinea-pigs after 16-17 hours of food deprivation is much lower than in those from the perirenal or epididymal depots of the same animals (21). Somehow, these adipocytes have not responded to the endocrine conditions of fasting, although as these data show, they are perfectly capable of large increases in lipolysis. The perinodal adipocytes are more sensitive to noradrenaline applied alone and in combination with tumour necrosis factor-a (TNFa) or interleukin-6 (IL-6), and their maximum rate of lipolysis is much higher than that of the nodeless depots, and significantly higher than that of adipocytes from elsewhere in node-containing depots.

Incubation with mixtures of cytokines and noradrenaline reveals even larger within-depot differences in the control of lipolysis. Adipocytes taken from sites within the same depot as little as 5 mm apart release glycerol at widely different rates under the same conditions (20). Figure 13.3 shows such data from the poplineal samples. Corresponding samples from the mesentery and omentum produce a similar picture. High doses of noradrenaline combined with 24 h of incubation with TNFa or IL-6 stimulated lipolysis, while other combinations of cytokines suppress the process to below the control values. These properties indicate that in the in vivo

Figure 13.2 Means + SE of spontaneous and noradrenaline-stimulated release of glycerol from adipocytes from near to lymph node(s) (dark bars) and far from lymph node(s) (light bars) on the first day of the experiment, without any prior treatment (21). Shaded bars: popliteal; horizontally striped bars: mesenteric; diagonally striped bars: omental; wavy bars: perirenal; n = 12 guinea-pigs, body mass 1096 +35g, age 16.0 + 0.2 months, fasted for 16-17 hours. Asterisks denote significant differences (Student's i-test) between pairs of samples from the same depot under the same conditions: *** significantly different at P< 0.001; ** significantly different at P<0.01; * significantly different at P < 0.05

Figure 13.2 Means + SE of spontaneous and noradrenaline-stimulated release of glycerol from adipocytes from near to lymph node(s) (dark bars) and far from lymph node(s) (light bars) on the first day of the experiment, without any prior treatment (21). Shaded bars: popliteal; horizontally striped bars: mesenteric; diagonally striped bars: omental; wavy bars: perirenal; n = 12 guinea-pigs, body mass 1096 +35g, age 16.0 + 0.2 months, fasted for 16-17 hours. Asterisks denote significant differences (Student's i-test) between pairs of samples from the same depot under the same conditions: *** significantly different at P< 0.001; ** significantly different at P<0.01; * significantly different at P < 0.05

situation, lymphoid cells could regulate lipolysis in adipocytes located in the vicinity of their node over a wide range of values and very precisely.

Human subcutaneous adipose tissue (presumably not associated with lymph nodes) releases small quantities of IL-6 (22), and cytokines from such sources may somehow be involved in the slow development of chronic disease (23). But in the short term, cytokines secreted in and around lymph nodes that 'leaked' into the bloodstream would have little effect on the large, nodeless depots that contain the great majority of the body's lipid stores: lipolysis in adipocytes from the perirenal and gonadal depots was unaltered by these mixtures of cytokines (21).

Noradrenaline also stimulates the smooth muscle of lymph vessels (24,25). The application of regular electrical pulses to the lumbar sympathetic ganglion produced a threefold increase in the flow of lymphocytes out of the popliteal ganglion of a sheep (26). This (and many other) lymph nodes are supplied by numerous very fine afferent lymph vessels that branch from the main afferent vessel and enter the node over almost its entire surface (27,28). Such tiny vessels are permeable to large molecules and even some kinds of small cells (29). Although the

Figure 13.3 The effect of pre-incubation with 10ng/mL IL-4 alone and with 0.5ng/mL interleukin-6, or 10ng/mL TNFa on spontaneous and noradrenaline-stimulated release of glycerol from adipocytes from near (darker bars) and far from (light bars) lymph nodes in the popliteal depot of the same guinea-pigs as for Figure 13.2 (21). All measurements were made on the second day post mortem. Asterisks denote significant differences from the corresponding sample incubated without cytokines: *** significantly different at P< 0.001; ** significantly different at P<0.01; * significantly different at P<0.05. For clarity, symbols indicating within-depot differences, and those indicating that all the values from 'near node' adipocytes are significantly different at P< 0.001 from those from the corresponding control samples incubated without cytokines, are not shown. Daggers denote significant differences between incubation with two cytokines and the corresponding sample incubated with IL-4 alone: ttt significantly different at P< 0.001; tt significantly different at P<0.01; t significantly different at P<0.05

Figure 13.3 The effect of pre-incubation with 10ng/mL IL-4 alone and with 0.5ng/mL interleukin-6, or 10ng/mL TNFa on spontaneous and noradrenaline-stimulated release of glycerol from adipocytes from near (darker bars) and far from (light bars) lymph nodes in the popliteal depot of the same guinea-pigs as for Figure 13.2 (21). All measurements were made on the second day post mortem. Asterisks denote significant differences from the corresponding sample incubated without cytokines: *** significantly different at P< 0.001; ** significantly different at P<0.01; * significantly different at P<0.05. For clarity, symbols indicating within-depot differences, and those indicating that all the values from 'near node' adipocytes are significantly different at P< 0.001 from those from the corresponding control samples incubated without cytokines, are not shown. Daggers denote significant differences between incubation with two cytokines and the corresponding sample incubated with IL-4 alone: ttt significantly different at P< 0.001; tt significantly different at P<0.01; t significantly different at P<0.05

authors of these studies do not mention the adipose tissue, the consequences of these anatomical arrangements and physiological properties in vivo would be to bring lymphoid cells and the adipo-cytes immediately surrounding the node into close proximity, enabling them to exchange metabolites.

The observations on multiple samples taken from large adult guinea-pigs summarized in Figures 13.1-13.3 highlight the limitations of conclusions based only on the perirenal or epididymal depots or on 3T3 adipocyte cell lines, from which no site-specific information can be obtained. In particular, they challenge the long-held assumption that all adipocytes in an anatomically defined depot respond equally to blood-borne and neural stimuli, and each adipocyte makes a small but equal contribution to the concentration of metabolites in the blood. The data in Figures 13.1 and 13.3 suggest that a small fraction of the total adipose mass responds strongly to cytokines, and the rest very little or not at all. In brief, most of the 'hard work' of responding rapidly to the fluctuating state of lymphoid cells in a lymph node is performed by a few adipocytes, while the others, which unfortunately are the ones most widely studied, respond more slowly to stronger and more persistent stimuli. This concept should be considered when comparing levels of blood metabolites with the properties of samples of adipocytes in vitro. Inappropriately chosen samples can sometimes produce misleading data (30).

In the popliteal depot of the rat, receptors for TNFa are much more adundant on the adipocytes that enclose the lymph node in a shell approximately 1mm ( = 10-15 adipocytes) thick (31). Type II (p75) receptors are continuously present on perinodal adipocytes, as well as on many of the lymphoid cells within it and endothelial cells. Type I (p60) appear on adipocytes surrounding the popliteal lymph node within 30 minutes of a stimulated immune challenge to the region of the lower leg drained by this node (Figure 13.4), and on the homologous adipocytes of the unchallenged leg within 24 h. These receptors cannot be seen on adipocytes elsewhere in the popliteal lymph node, although if the signal gets as far as the other leg, it presumably also reaches the rest of the adipose depot. On a longer time scale, this simulated immune stimulus also increases vascularization of the activated adipose tissue (32). These observations indicate that adipocytes around lymph nodes are

Figure 13.4 Immunofluorescent visualization of receptors for tumour necrosis factor-a on adipocytes around the popliteal depot of a rat. The field of a view is a little over 1 mm wide. (a) Bright-field view of a thick section (120 ^m) through the popliteal adipose depot and the lymph node enclosed therein (bottom right) that has been stained with FITC-labelled antibody to type II receptors for tumour necrosis factor-a. All the adipocytes appear similar. (b) The same section illuminated with ultraviolet light. The antibody binds to cells in the lymph node itself and to adipocytes surrounding it, but those more than 0.5 mm remote from the node remain unstained. The blood vessel visible as a nearly horizontal black line in (a) also picks up stain. (Courtesy of H. MacQueen(31))

Figure 13.4 Immunofluorescent visualization of receptors for tumour necrosis factor-a on adipocytes around the popliteal depot of a rat. The field of a view is a little over 1 mm wide. (a) Bright-field view of a thick section (120 ^m) through the popliteal adipose depot and the lymph node enclosed therein (bottom right) that has been stained with FITC-labelled antibody to type II receptors for tumour necrosis factor-a. All the adipocytes appear similar. (b) The same section illuminated with ultraviolet light. The antibody binds to cells in the lymph node itself and to adipocytes surrounding it, but those more than 0.5 mm remote from the node remain unstained. The blood vessel visible as a nearly horizontal black line in (a) also picks up stain. (Courtesy of H. MacQueen(31))

equipped to amplify their capacity to respond to lymphoid cells within a few hours of their activation.

This concept is confirmed by in vivo studies. When a single popliteal lymph node is activated by the long-established procedure of injecting a small quantity of lipopolysaccharide into the tissues that it drains, lipolysis in the adipocytes immediately surrounding it increases within an hour, and remains elevated for at least 9 hours before declining almost to baseline (33). Adipocytes thus activated also become more sensitive to noradrenaline, a synergism that suggests that the adipose tissue around the lymph nodes may be a forum for interactions between sympathetic stimulants such as stress and exercise, and immune function. These effects can be amplified simply by incubating excised adipose tissue explants in tissue culture medium for 24 h, strongly implicating paracrine and/ or auto-crine interactions in perpetuating the response to the immune stimulus after it has been removed from contact with the activated lymph node.

Cytokines generally seem to act locally in a paracrine or autocrine manner, with only small quantities reaching all organs via the general circulation (34). Paracrine interactions between adipocytes are becoming more widely recognized (35). There would be good reason for keeping cytokine-me-diated interactions between adipose tissue and lym-phoid cells local and transient. High levels in the blood cause severe malfunction of the lungs, kidneys and other vital organs, leading to septic shock syndrome. Moderate blood levels of this cytokine for long periods are associated with abrupt, sustained depletion of adipose tissue lipids and muscle wasting, leading to cachexia, a common complication of cancer and chronic bacterial disease, and possibly at lower concentration to insulin resistance (30).

To find out more about what lymph node lym-phoid cells might be getting by stimulating lipolysis in the adipose tissue around them, we compared the fatty acid composition of triacylglycerols in adipose tissue from different parts of depots that contain lymph nodes (Figure 13.5) (5). In all those examined, but especially in the intermuscular, omental and mesenteric depots, there were fewer saturated fatty acids, and more polyunsaturates in the triacyl-glycerols found in the adipose tissue 1-2mm around the nodes than elsewhere in the depot.

The adipose tissue from around lymph nodes that in vitro interacts most strongly with lymphoid cells, and has the largest responses to TNFa and the interleukins, also contains a greater proportion of the very fatty acids that these cells need for their proliferation and integrated function, and cannot make for themselves. Selective release and retention of certain fatty acids has been demonstrated in adipocytes in vitro (36,37), suggesting how such site-specific differences could arise. The processes measured in Figures 13.2-13.4 suggest some reasons why they exist: selective, local stimulation of lipolysis from the adipocytes near the nodes would maximize supplies of polyunsaturated fatty acids to the activated lymphoid cells. Lipolysis from these adipocytes is not strongly stimulated by fasting (Figure 13.2), so these local controls determine fatty acid release regardless of fever, anorexia or other whole body state that the larger 'general storage' depots (e.g. perirenal, inguinal) readily respond to. These observations are also consistent with the reports that lymphocyte function is more strongly modulated by polyunsaturated fatty acids than by monounsaturates or saturates both in vitro (38) and in vivo (39).

While many of the fatty acids so released were probably oxidized, some would have been incorporated into membrane phospholipid and/or serve as precursors for lipid-based messenger molecules for the proliferating lymphocytes. The increase in proportion of polyunsaturated fatty acids in rat liver lipids following 10 days of chronic infusion of TNFa has been attributed to changes in liver metabolism (40). But such 'new' fatty acids could equally come from triacylglycerols in the adipose tissue around lymph nodes, in which lipolysis is especially sensitive to this cytokine (21), and poly-unsaturated fatty acids are more abundant (Figure 13.5). This concept of local provision of fatty acids should be considered for investigations into effects of diet on lipids in lymphoid tissue (41), and the relationship between dietary lipids, adipocyte composition and breast cancer (42).

Certain adipose depots also have significant capacity for the synthesis and release of glutamine (3), that activated lymphoid cells use in large quantities (43). Provision of glutamine to support protein synthesis in lymphoid cells may be another way in which adipose tissue supplies the immune system during periods of anorexia and cachexia, when external sources are greatly reduced, and competition with other tissues such as muscle may be strong.

Such site-specific differences in the composition of the storage lipids came as a surprise—previous investigators had assumed that continuous lipolysis and re-esterification of triacylglycerols would eventually homogenize the entire store. The only other example of site-specific differences in fatty acid composition of triacylglycerols hitherto described were the extremities and superficial adipose tissue of some cold-adapted mammals (12,44) which, although similar in principle, differ in some important details. The adaptations of adipose tissue triacyl-

Figure 13.5 Means+ SE of the proportions of saturated FAs, monounsaturated FAs, linoleic acid (18:2n-6) and a-linolenic acid (18:3n-3) extracted from the triacylglycerols in samples of adipose tisue from six sites in the popliteal depot and four sites in the intermuscular cervical depot between the neck muscles (5). Popliteal samples 1 and 2 were from as near as possible to the node on the distal and proximal sides; 3 and 4 from the middle of the depot near where the sciatic nerve runs through it towards the gastrocnemius muscle, respectively about 4 mm and 6 mm anterior to the node; sample 5 was from as far as possible from the node going towards the anterior, behind the knee joint; sample 6 was from as far as possible from the node going dorsally. Cervical sample 1 was from near the large central node; 2 near the group of smaller nodes near the dorsal edge of the adipose depot; 3 and 4 from opposite sides of the depot, as far away as possible from lymph nodes. n = 17 adult guinea-pigs fed on plain chow. Asterisks refer to differences between the composition of sample 1 and others from the same depot, assessed using Student's i-test: *** Significantly different at P< 0.001; ** significantly different at P<0.01; * significantly different at P<0.05

Figure 13.5 Means+ SE of the proportions of saturated FAs, monounsaturated FAs, linoleic acid (18:2n-6) and a-linolenic acid (18:3n-3) extracted from the triacylglycerols in samples of adipose tisue from six sites in the popliteal depot and four sites in the intermuscular cervical depot between the neck muscles (5). Popliteal samples 1 and 2 were from as near as possible to the node on the distal and proximal sides; 3 and 4 from the middle of the depot near where the sciatic nerve runs through it towards the gastrocnemius muscle, respectively about 4 mm and 6 mm anterior to the node; sample 5 was from as far as possible from the node going towards the anterior, behind the knee joint; sample 6 was from as far as possible from the node going dorsally. Cervical sample 1 was from near the large central node; 2 near the group of smaller nodes near the dorsal edge of the adipose depot; 3 and 4 from opposite sides of the depot, as far away as possible from lymph nodes. n = 17 adult guinea-pigs fed on plain chow. Asterisks refer to differences between the composition of sample 1 and others from the same depot, assessed using Student's i-test: *** Significantly different at P< 0.001; ** significantly different at P<0.01; * significantly different at P<0.05

glycerols to cooler conditions mainly involve substituting saturated fatty acids with monounsatu-rates. In this case (Figure 13.5), the saturates decrease as the relative abundance of the poly-unsaturates increase, with the proportions of mono-unsaturates remaining constant.

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