Local immune responses in afferent and efferent lymph

Acquired immune responses are dynamic processes dependent upon the co-ordinated interaction of antigen-trapping accessory cells and lymphocytes of the immune system in specialized secondary lymphoid tissues that include lymph nodes, Peyer’s patches and spleen. The function of the immune system is to clear infections or neoplastic changes locally wherever they appear while avoiding autoimmune responses and non-specific damage of unaffected tissues. Mature lymphocytes with a broad range of potential antigen specificities that are not autoimmune are generated in the primary lymphoid tissues. Many, if not all, of these circulate between the blood and the tissues by way of the lymph nodes. The majority of small lymphocytes recirculate between the blood and secondary lymphoid tissue either directly through post-capillary venules or indirectly via tissues such as the skin and the gut mucosa.1–5 Blood CD4+ and CD8+ T cells recirculate efficiently, whereas a subset of B cells and γδ T cells do not.6–8 T and B cells (particularly CD4+ T cells) from peripheral lymph nodes and from gut-associated lymphoid tissue tend to recirculate to the tissue of origin,3–5, 9 although this tissue-specific recirculation is by no means absolute. Recirculating lymphocytes can, therefore, monitor tissues for the presence of foreign antigens and enhance the recruitment of antigen-specific cells to the local lymph node. This tissue-specific immune surveillance by lymphocytes is facilitated by a network of lymph nodes distributed strategically around the body and connected by a system of lymphatic vessels. The purpose of this article is to review recent advances in our understanding of the dynamics of local immune cell and cytokine responses in the two strikingly different compartments of afferent and efferent lymph that, respectively, enter and exit peripheral lymph nodes. The emphasis will be on studies in afferent and efferent lymph entering and exiting skin-draining lymph nodes in ruminants where lymphatic cannulation is relatively easy to perform. Afferent lymphatics are blind-ended vessels that arise in and permeate the tissues and selectively convey lymphocytes and antigen-presenting accessory cells into the subcapsular and medullary sinuses of lymph nodes. Efferent lymphatics (usually only one or two for each node) carry lymphocytes and lymph plasma out of lymph nodes ultimately into the major efferent ducts and then back to the blood. Efferent lymph from regional lymph nodes may pass through one or more lymph nodes downstream before reaching a major efferent duct. Under physiological conditions, there are profound differences between afferent and efferent lymph flow rates, cell content and activation status of the cells (Fig. 1, Table 1) Flow rates and cellular composition of lymph around single skin-draining lymph nodes of adult sheep. Afferent lymph contains antigen-presenting dendritic cells, T cells and a small proportion of B cells. Lymph flow and cell output rates are low. Efferent lymph contains >98% lymphocytes with a higher proportion of B cells and much higher flow and cell output rates than in afferent lymph.2–5 Greater than 90% of efferent lymphocytes are derived from the blood via high endothelial venules (HEV). L-selectin on lymphocytes is associated with adhesion to peripheral lymph node HEV. For the details of the cytokines studied to date, see Fig. 2 and the text. Analysis of afferent lymph gives an accurate picture of immune and inflammatory responses in the tissues as they occur. Afferent lymph contains antigen-presenting dendritic cells (ALDC), T cells (principally CD4+ T cells) and a small proportion of B cells, all migrating with a low cell output from the tissues into the local lymph node3, 5, 10–12 (Fig. 1, Table 1). This is consistent with continuous immune surveillance, involving dendritic cells and CD4+ T cells in particular, but with a lesser involvement of B cells. γδ receptor+ T cells (γδ T cells) are prevalent in the blood of ruminants11, 12 and are the most abundant lymphocytes in normal ruminant skin (Table 1). However, they comprise a small population in afferent lymph compared to αβ T cells (Table 1) which suggests that they reside in the tissues longer than αβ T and B cells, and raises the possibility that a proportion of them may die there unless stimulated. This would be consistent with their envisaged role as surveillance cells for epithelial surfaces, with a lesser tendency to recirculate than αβ T cells.12 Afferent lymph T cells exhibit an activated phenotype compared to efferent lymph cells, with a higher proportion of T cells expressing interleukin (IL)-2Rα (CD25) and major histocompatibility complex (MHC) class II DQ antigens (Table 1). CD4+ T cells in ‘resting’ afferent lymph also express mRNA for the cytokines IL-2, IL-4, IL-6, IL-10 and interferon-γ (IFN-γ) (Fig. 3 and unpublished). This activated phenotype may be important for the migration of the T cells through the tissues and into afferent lymph. Afferent lymph plasma under physiological conditions contains the cytokines IL-1β, tumour necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-8,13, 14 and other biological activities generated in the tissue of origin.3, 13–15 These cytokines can potentially influence cellular responses within the lymph node. Afferent lymph is important in maintaining lymph node integrity and architecture,16 and the cytokines/mediators and/or the afferent cells that they support are candidates for this function. These include the cytokines TNF-α and lymphotoxin (TNF-β) which are associated with the development and maintenance of lymph nodes and lymphoid architecture.17–19 2P-labelled cytokine-specific oligonucleotides (internal sequence). The results show that activated CD4+ T cells exhibiting type 1 (IFN-γ) and type 2 (IL-4, IL-6 and IL-10) cytokine expression are concentrated in afferent lymph. Efferent lymph contains >99% lymphocytes with a high proportion of CD4+ T cells and B cells compared to CD8+ and γδ T cells. Lymph flow and cell output is high (Fig. 1, Table 1). Greater than 90% of lymphocytes in efferent lymph are derived from the blood via high endothelial venules (HEV) and less than 4% are generated within the node under normal physiological conditions.2 Greater than 90% of lymphocytes enter the lymph node by HEV as compared with <10% from afferent lymph,3–5 demonstrating that the predominant recirculation route in the periphery for small lymphocytes is from the blood to the lymph nodes. The majority of efferent lymphocytes differ from afferent lymphocytes in not having an activated phenotype (Table 1 and Fig. 2). Efferent lymph plasma drains from within the lymph node, but cytokine levels, with the exception of GM-CSF, are undetectable or very low. 1 The results show a marked concentration of activated cytokine-producing cells in afferent lymph compared to efferent lymph during the anamnestic immune response, consistent with an activated effector/memory cell population in afferent lymph. A function for IL-8 (which precedes IL-1β detection14) in the initial phase of the anamnestic immune/inflammatory response is also implied. Recent studies have shown that peripheral blood B cells in sheep consist of two subpopulations that differ in their capacity to recirculate through lymph.6–8 The recirculating subpopulation has the phenotype CD21+, CD11b−, L-selectin+ and is detected in afferent and efferent lymph and lymph nodes. The second subpopulation comprises ≈30–50% of blood-borne B cells, has the phenotype CD21−, CD11b+, L-selectin− and shows enhanced migration to the spleen. The significance of these phenotypes and their distribution is not known, but the presence of the two subpopulations may represent different stages of B cell activation or a division into ‘naïve’ and ‘memory’ cells as described for T cells. Kimpton and colleagues have shown that the recirculation of the different lymphocyte subsets (non-random T and B distribution) is the same in fetal lambs (which are immunologically virgin) as in adult animals. This demonstrates that the pathways of lymphocyte recirculation are imprinted during fetal life in the absence of antigen and remain stable through adult life.20 During immune responses, profound changes take place in the cellular composition and activation status of the cells in afferent and efferent lymph. The gross changes are shown in Fig. 1, and examples of changes in cytokine production following antigen challenge are shown in Fig. 2. Following antigen challenge or infection of the tissues, there is an early (and usually transient) inflammatory response during which neutrophils can be detected in the afferent lymph.3 In sheep cutaneously challenged with the larvae of Lucilia cuprina (the cause of blowfly strike), or the parapoxvirus orf virus, rapid increases in afferent lymph plasma levels of the pro-inflammatory cytokines IL-1β, TNF-α, and the chemokine IL-8 were detected concomitant with the appearance of neutrophils in the lymph.13, 14 Following this phase there is an increase in the frequency of lymphoblasts, particularly CD4+ T-cell blasts, and in sheep reinfected with orf virus these cells are the principal source of the lymphokines IL-2, IFN-γ, and GM-CSF (ref. 14 and unpublished; Fig. 2). As CD4+ T cells are the principal source of regulatory cytokines in the antigen-specific phase of immune responses, this might have the effect of providing the appropriate tissue environment in which other lymphocytes and inflammatory cells could operate to clear antigen or infectious agents. In primary immune responses in afferent lymph, the appearance of lymphoblasts is delayed compared to anamnestic responses. Furthermore, antibodies are detected during anamnestic responses. In spite of all these changes, in most studies afferent lymph flow rate and cell output did not increase significantly during either primary or anamnestic immune responses. The exception to this is in sheep reinfected with orf virus14 or challenged with orf virus antigen (Fig. 2) where cell output can triple. Changes in dendritic cells are discussed below. In efferent lymph, three phases in the lymph response to antigen or infection have been described.2–5 Firstly, there is often a ‘shut down’ of lymphocyte output, which lasts for up to 24 hr. Secondly, there is a subsequent rise in efferent lymphocyte output (‘recruitment phase’) associated with increased blood flow to the node and thought to represent an increased influx of lymphocytes across HEV for rapid screening of antigen on APC within the node. Efferent lymphoblasts appear after this phase. These are effector T and B cells and do not recirculate. The lymph node is the principal site of B-cell blast, plasma cell and antibody production, all of which are concentrated in efferent as opposed to afferent lymph. Thirdly there is the resolution phase, the onset of which is dependent upon the continuing presence of antigen/pathogen in the tissues and during which lymph flow rate and cellular composition return to pre-stimulation levels. Shutdown and recruitment are particularly marked during anamnestic immune responses and resolution is more rapid than in primary responses. Following a first or second encounter with antigen or a pathogen, a sequential appearance of CD4+ T cells followed by CD8+ T cells and/or B cells has been described in efferent lymph.5, 9, 21, 22 This may represent different transit times through the node where CD4+ T cells are selected most rapidly.5, 22 This could be to expedite their antigen-specific activation in order to provide cognate help and IL-222 (or other lymphokines) for CD8+ T- and B-cell activation and subsequent migration. Lymphoblasts exit the node in the same phenotypic sequence, and in sheep reinfected with orf virus, CD4+ and CD8+ T-cell blasts are the source of the lymphokines IL-2, GM-CSF and IFN-γ.21 The production of lymphokines by efferent lymph cells (using a cell to cell comparison) is low compared to that of afferent lymph cells14, 21 ( Figs 2 and 3). This is probably because only a small number of efferent lymph T cells are activated by antigen and cytokines within the node. These are effector cells subsequently recruited to the tissues. A similar sequential output of lymphocytes and lymphoblasts has been described in afferent lymph during orf virus reinfection in sheep.14 Whether CD4+, CD8+, γδ T- or B-cell output predominates in either lymph compartment depends on the antigen or infectious agent, and demonstrates the usefulness of lymphatic cannulation for studying the type of immune response generated by different pathogens.5, 9, 23 For example, in sheep reinfected with orf virus, CD4+ T cells predominate at all stages of infection,14, 21 whereas in sheep infected with Toxoplasma gondii CD8+ T cells predominate in the later stages of infection,24 and in sheep infected with Salmonella abortus ovis B cells predominate.25 A seminal study in sheep showed that T cells that were naïve with respect to antigen encounter (measured by CD45RA expression and low levels of a range of adhesion molecules) predominated in efferent lymph.26 This indicated that they had recirculated from the blood into lymph nodes via HEV where antigen-reactive cells clonally expand in number. Memory T cells (lacking CD45RA expression and exhibiting high levels of a range of adhesion molecules) predominated in afferent lymph indicating that they had recirculated by way of the tissues where they can react rapidly to recall antigens precisely where they penetrate the body’s innate defences.26 Westerman and Pabst have argued that this scheme may be premature, based upon a critical evaluation of more recent data.27 In fact, adult sheep popliteal and prefemoral efferent lymph contain an ≈1:1 ratio of CD45R+ and CD45R− T cells21 and not the >90% CD45R+ dominance described previously26 (Table 1). This indicates that the putative ‘memory/effector’ phenotype cells can cross HEV into peripheral lymph nodes. A recent study in rats led to the same conclusion.28 It is clear from studies in rodents that the division of naïve and memory cells based on the expression only of CD45R isoforms is inaccurate and does not reflect a dynamic change in expression of these molecules following activation.28–30 Furthermore, in antigen-naïve fetal lambs (where the placentation does not allow external antigens access to the fetus), both CD45RA+ and CD45RA− T cells were found in fetal blood and lymph, with enhanced migration of both T-cell subpopulations through the tissues and in afferent lymph compared to adult animals.20 This demonstrates that genuinely naïve T cells are either CD45RA+ or CD45RA− in the ovine fetus. L-selectin expression appears to be necessary for lymphocyte recirculation through peripheral lymphoid tissue,31 whereas other integrins are expressed by lymphocytes recirculating through other tissues, for example integrin β7 for intestinal tissues.32 The lack of CD45RA (sheep) or expression of CD45RO (cattle) in lymph may be more useful as markers of activated or effector T cells. Following orf virus antigen challenge or reinfection in sheep, CD4+ and CD8+, CD45RA− T cells were the principal source of IL-2, IFN-γ and GM-CSF in both afferent lymph (which does not contain CD45RA+ T cells) and efferent lymph14, 21 (Fig. 2). In cattle, CD4+/CD45RO+ but not CD4+/CD45RO− T cells proliferate and produce multiple lymphokines rapidly after stimulation in culture.33 Orf virus-specific CD8+ cytotoxic T lymphocytes (CTL) that are directly cytotoxic for orf virus-infected autologous target cells (effector as opposed to precursor CTL) are also enriched in afferent lymph (and lack CD45RA) compared to efferent lymph.34 However, the proportion of cells expressing the CTL activation enzyme N-α-benzyloxy-carbonyl–lysine thiobenyl ester (BLT)-esterase is greatly reduced in afferent lymph compared to the infection skin site. This indicates that the activated cells are predominantly retained at the site of infection where they are required and do not migrate in large numbers to cause non-specific damage in adjacent tissues (ref. 34 and unpublished). Further work is required to define and map naïve, memory and effector cells as well as the function of the CD45R isoforms before the issue of naïve/memory T-cell phenotype and distribution can be resolved Dendritic cells are potent antigen-presenting cells that initiate primary and anamnestic immune responses. Studies in mouse and man have shown that there is a maturational pathway for dendritic cells in the skin, from immature antigen-capturing epidermal Langerhans’ cells to cells which migrate and mature into potent antigen-presenting dendritic cells in the dermis and paracortical areas of lymph nodes.35–37 Skin-draining afferent lymph dendritic or veiled cells (ALDC) represent a migrating population of dendritic cells but not to epidermal Langerhans’ cells and lymph node These migrating cells are not studied in mouse and antigen from the tissues to the paracortical areas of lymph nodes and are potent antigen-presenting cells for T cells in primary and anamnestic immune with their antigen-presenting ruminant express high levels of class and II the molecules and and and a range of adhesion molecules and The expression of and on ruminant and is associated with that is an in antigen A proportion of ovine are in express and a proportion of also express (the which on these cells the to which have been shown to be in antibody responses via interaction with B These phenotypes provide with means of and and antigens to initiate both primary and anamnestic immune responses. A small proportion of the migrating are thought to as cells in the paracortical areas of lymph nodes where they can with T cells and initiate (and immune The between and in the of lymph nodes is not antigen in a for T cells whereas may lymph antigen for B-cell In as in mouse and the exhibit phenotypic and In cattle, two of have been with expression of to differences in antigen or of T In different the cytokines TNF-α, GM-CSF, IL-1β, IL-4, IFN-γ and IL-10 in particular, appear either to Langerhans’ cell or to of dendritic cell antigen and to T cells. For example, TNF-α the of dendritic IL-1β and GM-CSF the expression of dendritic cell adhesion and molecules whereas IL-10 and IFN-γ However, the role of the different cytokines is to in rodents and as the studies are by the of different dendritic cells at different stages of derived from either or from the tissues. afferent lymph on the other a source of antigen-presenting dendritic cells for which are or cell and/or In studies with the pro-inflammatory cytokines IL-1β and TNF-α while the cytokines IL-10 and In sheep, TNF-α and (and are expressed in the skin and afferent and are to dendritic cell and function following antigen in with TNF-α and increase in number if GM-CSF is also IFN-γ this TNF-α and GM-CSF into ovine skin the recruitment of Langerhans’ cells and these results that the cytokines are of the and accessory function of dendritic cells in the skin and afferent lymph. The of afferent lymph an to the cytokine of dendritic cell phenotypic and that is a of in rodents and may the T and B cells into the node via the afferent lymph. between and T cells for T-cell activation have been and In primary immune responses, that have antigen by and/or or via but stimulation of T cells in the tissues is because of the lack of antigen-specific T cells. However, will antigen with to the lymph node where large numbers of lymphocytes can be for low cells, after which and can followed by of antigen-specific T cells. In anamnestic responses, enhanced via by will interaction with memory CD4+ T cells to rapid production of T and B effector cells. of with T cells and inflammatory cytokines to the node of the appropriate anamnestic response that place by recruitment of antigen-specific lymphocytes from the blood. The and of molecules on and T may levels will a effector T-cell response whereas low levels will to no and T-cell and in the lymph node. Recent studies in rodents that differences in the of antigen stimulation may be required to naïve T-cell and may the cells proliferate or by the pathway the for T-cell the number of molecules to be by and cell or T-cell The lack of mRNA in γδ T indicates different activation and for these cells compared to αβ T upon interaction with their has with that γδ T cells might to molecules and by Dendritic cells do not appear in efferent lymph and and die in the lymph node. is a which has been shown in and which have not with T therefore, have not as antigen-presenting cells. that have antigen in the lymph node are also rapidly Furthermore, if express of they may be by cytotoxic T Studies on immune responses in afferent and efferent lymph of lymph nodes the tissues have to the that primary immune responses are generated in the lymph nodes with a before antigen-specific effector cells at the memory cells are there is the possibility of an but local anamnestic response in the tissues. This would memory cells recruited from the by a more tissue influx of antigen-specific lymphocytes from the lymph node which is the site of effector T- and B-cell during anamnestic immune responses. These studies also the of dendritic cells in immune surveillance of the tissues, of primary immune responses, and interaction with putative effector T cells T cells in of the lymph node during anamnestic responses. The interaction of T cells with dendritic cells in the tissues as well as within lymph nodes may be important not only in immune responses but also in peripheral is no division of type 1 and type 2 immune responses in although and colleagues have that a type environment in the tissues and a type environment in the skin may tissue-specific migration of lymphocytes Recent studies in rodents and man cytokine production to cells in and that T 1 cells produce IL-2, IFN-γ, TNF-α, are more detected than cells (IL-4, IL-6, in the tissues, in In sheep, IFN-γ mRNA is detected more than mRNA in efferent lymph T cells during challenge and type 2 antibody is detected in prefemoral efferent lymph as well as in skin In T cells migrating from the skin contain for IL-4, IL-6 and IL-10 as well as IFN-γ (Fig. 3). Afferent lymph and the cells contains may a role with adhesion molecules in maintaining lymphocyte recirculation as well as antigen for primary and secondary immune responses and maintaining the integrity of lymph nodes. The presence of activated cells and cytokines in afferent lymph under physiological conditions would support The dendritic cell might lymphocytes for migration in afferent lymph through as well as the type of immune response generated in the tissues. The expression of adhesion on tissue the recruitment of lymphocytes expressing appropriate tissue-specific molecules in response to Dendritic cells that are and to the tissues in which they reside may different of antigens to the recruited T cells. For example could be for These can and the effector phenotype of T cells through the expression of molecules and cytokines for This does not the involvement of other antigen-presenting cells during anamnestic responses. are that can be in the to lymphatic of which provide for