Bailey, S.

Gossner, Anton

Hopkins, John

Hunter, Nora

English

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a 'memory' phenotype, being CD62L(-)/CD45RA(-) and expressing high levels of CD2 and CD11a; efferent cells are largely 'naïve', being CD62L(+)/CD45RA(+) with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNgamma, TGFbeta and TNFalpha) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments

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     Edinburgh Research Explorer                                      Patterns of cytokine gene expression of naïve and memory Tlymphocytes in vivo.Citation for published version:Gossner, A, Bailey, S, Hunter, N & Hopkins, J 2002, 'Patterns of cytokine gene expression of naïve andmemory T lymphocytes in vivo.' Veterinary Immunology and Immunopathology, vol 10, no. 87, pp. 261-264.Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher final version (usually the publisher pdf)Published In:Veterinary Immunology and ImmunopathologyPublisher Rights Statement:Copyright 2002 Elsevier ScienceGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015Patterns of cytokine gene expression of naı¨ve andmemory T lymphocytes in vivoAnton G. Gossnera, Samantha Baileya, Nora Hunterb, John Hopkinsa,*aLaboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UKbIAH Neuropathogenesis Unit, King’s Buildings, West Mains Road, Edinburgh, UKAbstractLarge-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes viaafferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferentlymphocytes have distinct phenotypes; afferent lymphocytes have a ‘memory’ phenotype, being CD62L/CD45RA andexpressing high levels of CD2 and CD11a; efferent cells are largely ‘naı¨ve’, being CD62Lþ/CD45RAþwith low levels of CD2and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated invitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can bestimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobeRNAase protection assay for 13 sheep cytokines (IL-1b, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNg, TGFband TNFa) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expressionof cytokine mRNA in MACS-purified CD4þ and CD8þ T lymphocytes from both lymphoid compartments.# 2002 ElsevierScience B.V. All rights reserved.Keywords: Cytokines; T lymphocytes; Memory figure1. IntroductionThe physiological processes of the immune system,especially adaptive immune responses, are dynamicprocesses dependent upon the co-ordinated interactionof antigen-trapping accessory cells and lymphocytesin specialised peripheral lymphoid organs such aslymph nodes. The patterns of lymphocyte recircula-tion through different immunological compartmentshave been well defined in the sheep. Blast cells fromperipheral nodes home to the spleen and lymph nodes,and within this small recirculating lymphocyte poolthere are subsets which preferentially recirculate thor-ough the skin, peripheral nodes or mucosal associatedlymphoid tissue (Mackay et al., 1988; Abernethy et al.,1991). The afferent lymph selectively conveys Tlymphocytes, a small proportion of B lymphocytes,afferent lymph dendritic cells (ALDCs) and macro-phages (Hall and Morris, 1965; Smith et al., 1970).The ALDCs are professional APCs involved in thecarriage of antigen in an immunogenic form from theskin to the draining lymph node.Efferent lymph contains only lymphocytes that enterthe lymph node, either from the blood or the afferentlymphatics of the node and leave via the efferent,lymphatics within the lymph plasma, ultimately backinto the blood (Hall and Morris, 1962; Smith et al.,1970). The lymph node is a central focus of the immuneVeterinary Immunology and Immunopathology 87 (2002) 261–264* Corresponding author. Tel.: þ44-131-650-6169;fax: þ44-131-650-6511.E-mail address: john.hopkins@ed.ac.uk (J. Hopkins).0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 5 0 - 8system and the use of cannulation of the afferentand efferent lymphatics provides a unique approachto the in vivo analysis of immune cell populations withinthe different immunological microenvironments ofsingle lymph nodes in the sheep. There are considerablephenotypic and functional differences between thecells in blood and afferent lymph suggesting that thereis selectivity in the extravasation of cells from the bloodacross the endothelium into the peripheral tissues.Emigration of afferent and efferent T lymphocytes,with their distinct phenotypes and function, can bedemonstrated by the circulation of naı¨ve and memoryT lymphocytes within an individual animal (Mackayet al., 1996).Fig. 1. Proliferation of CD4þ lymphocytes from both afferent and efferent lymph in vitro. In vitro proliferation assays of CD4þ T cells fromafferent (A and B) or efferent (C and D) lymph stimulated with 25 mg/ml ovalbumin (A and C) or allogeneic APC (B and D). 5 104lymphocytes were cultured with irradiated afferent DC (DC) or mammary macrophages (Mf) for 5 days and then labelled with 3H-thymidinefor 5 h.262 A.G. Gossner et al. / Veterinary Immunology and Immunopathology 87 (2002) 261–264The blood contains T lymphocytes of both naı¨veand memory phenotypes though afferent lymph Tlymphocytes are mainly of the memory cell phenotypeand efferent T lymphocytes draining the lymph nodeare mainly of a naı¨ve phenotype. Mature lymphocytes,both CD4þ and CD8þ, recirculate between the bloodand the tissues via the lymph nodes. Immunologicalmemory is disseminated from the node via the efferentlymph, where exit of T lymphocytes following antigenstimulation is non-random despite the fact that cellsfrom the efferent lymph have a ‘naı¨ve’ phenotype.A total of 90% of the lymphocytes within a lymphnode are derived from the blood, the remainder arefrom lymphocyte proliferation within the node andafferent lymph (Hall and Morris, 1965). CD4þ cellsare selectively enriched over CD8þ T lymphocytes inlymph nodes.To investigate the relationships between functionalactivities of lymphocytes and their migratory proper-ties, we have developed a multiprobe RNAase protec-tion assay (RPA) for 13 sheep cytokines (IL-1b, IL-2,IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF,IFNg, TGFb and TNFa), as well as two housekeepinggenes (ATPase and GADPH). Cannulation of theprefemoral ‘‘pseudo-afferent’’ and efferent lymphaticsof sheep allows the isolation of ‘resting’ traffickinglymphoid cells to be analysed for their constitutivecytokine mRNA expression.2. Naı¨ve, memory and effector lymphocytesAfferent lymph contains ALDCs, T lymphocytesand B lymphocytes, all migrating from the tissues tothe local lymph node. The afferent T lymphocytesexpress an activated phenotype (CD2HI CD58HICD44HI CD11aHI, high level expression of MHC IIDQþ and CD25) and high levels of adhesion mole-cules but are CD45RA compared to efferent lymphcells (Mackay et al., 1990, 1992; Abitorabi et al.,1996). In terms of function they respond to solubleantigen (Fig. 1A) and alloantigen presented by bothALDCs and macrophages (Fig. 1B).Efferent lymph contains >99% lymphocytes with ahigh proportion of CD4þ T lymphocytes and Blymphocytes compared to CD8þ and gd T lympho-cytes. T lymphocytes isolated from efferent lymph(CD2LO CD58LO CD44LO CD11aLO, class II MHCDQ and CD25) are CD45RAþ and express lowlevels of adhesion molecules (Mackay et al., 1990,1992; Abitorabi et al., 1996). These T lymphocytes arehighly responsive to both soluble antigen and alloanti-gen when presented by ALDCs. They respond veryweakly to soluble antigen presented by macrophagesand are non-responsive to allogenic macrophages in amixed leukocyte reaction (Fig. 1D).Using MACS-purified CD4þ and CD8þ T lym-phocytes from afferent and efferent lymph, the con-stitutive expression of cytokine mRNA in both cellpopulations using a multiprobe RPA was analysed.Preliminary results identified T lymphocytes from‘‘resting’’ afferent lymph that expressed mRNA forthe cytokines IL-1b, IL-3, IL-8, IFNg, TNFa and TGFbusing this assay system (Fig. 2). With T lymphocytesFig. 2. Measurement of cytokine mRNA levels using an RNAaseprotection assay. The RNAase protection assay is a sensitive andspecific method for the quantitation of mRNA species. The probetemplates were generated by RNA polymerase directed synthesis of32P-labelled anti-sense RNA from a cDNA template. The purified-labelled probes were hybridised with the target RNA isolated fromboth afferent (lanes 9–15) and efferent (lanes 3–8) lymph cells.After hybridisation was completed, ribonucleases specific forssRNA digested unhybridised RNA and probe. The ‘‘RNAase-protected’’ probes and undigested probe markers (lanes 1 and 2)were resolved on a denaturing PAGE and were visualised byphosphor imaging. Probe set 1* was used with the samples in lanes3–5 and 9–12. Probe set 2 was used with the samples in lanes 6–8and 13–15.A.G. Gossner et al. / Veterinary Immunology and Immunopathology 87 (2002) 261–264 263from ‘‘resting’’ efferent lymph expressed mRNA forthe cytokines IL-2, IL-3, IFNg, and TNFa weredetected (Fig. 2).3. Discussion‘Resting’ efferent lymph T lymphocytes with theirnaı¨ve phenotype require activation by ALDCs torespond to soluble antigen, yet ‘resting’ afferentlymph T lymphocytes do not require ALDCs foractivation, as they are activated by macrophages,though they still also respond to ALDCs. This clearlydemonstrates that afferent lymph responder cells have‘immunological memory’, thus enabling them torespond to antigen presentation by macrophages. Con-versely with ‘resting’ efferent lymph, there are fewunprimed responder cells able to respond to solubleantigen without stimulation by ALDCs. Thus afferentlymph T lymphocytes demonstrate a lower activationthreshold, i.e. ‘memory’, which is functionally illu-strated by their ability to respond both to ALDCs andmammary macrophages. In contrast efferent lymphT lymphocytes have a higher activation threshold,confirmed by their ability to respond to ALDCs butnot macrophages. Both sets of lymphocytes act inresponse to stimulation from ALDCs and yet havedistinctly different phenotypes.The responder cells of efferent lymph with theirnaı¨ve phenotype have recirculated from the blood buthave no ‘immunological memory’ and a higher acti-vation threshold, which is only overcome by presenta-tion of both soluble antigen and alloantigen by ALDCsand not macrophages. ‘Naı¨ve’ T lymphocytes travel toareas of secondary lymphoid organs in search ofantigen presented by dendritic cells. T lymphocytememory is demonstrated by afferent lymph cellsability to proliferate in response to soluble antigenpresentation by macrophages but the presence ofmemory effector cells is demonstrated by their abilityto respond to alloantigen presentation by both ALDCsand macrophages though to a lesser extent than withsoluble antigen.Further work needs to be done to map and definenaı¨ve, memory and effector cells before the issue ofnaı¨ve and memory phenotype and distribution can beresolved. These new RPA multiprobe sets providesvaluable tools for the simultaneous quantitative deter-mination of gene expression of multiple ovine cyto-kines of both constitutive and inducible type.AcknowledgementsSupport for the research described in this articlewas provided by a BBSRC research grant no. 15/BS410569.ReferencesAbernethy, N.J., Hay, J.B., Kimpton, W.G., Washington, E., Cahill,R.N.P., 1991. Lymphocyte subset-specific and tissue-specificlymphocyte–endothelial cell recognition mechanisms indepen-dently direct the recirculation of lymphocytes from blood tolymph in sheep. Immunology 72, 239.Abitorabi, M.A., Mackay, C.R., Jerome, E.H., Osorio, O., Butcher,E.C., Erle, D.J., 1996. Differential expression of homingmolecules on recirculating lymphocytes from sheep gut,peripheral, and lung lymph. J. Immunol. 156, 3111.Hall, J.G., Morris, B., 1962. The output of cells in lymph from thepopliteal node of sheep. Q. J. Exp. Physiol. 47, 360.Hall, J.G., Morris, B., 1965. The origin of the cells in the efferentlymph from a single lymph node. J. Exp. Med. 121, 901.Mackay, C.R., Kimpton, W.G., Brandon, M.R., Cahill, R.N.P.,1988. Lymphocyte subsets show marked differences in theirdistribution between blood and the afferent and efferent lymphof peripheral lymph nodes. J. Exp. Med. 167, 1755.Mackay, C.R., Marston, W.L., Dudler, L., 1990. Naive and memoryT cells show distinct pathways of lymphocyte recirculation. J.Exp. Med. 171, 801.Mackay, C.R., Marston, W.L., Dudler, L., Spertini, O., Tedder, T.F.,Hein, W.R., 1992. Tissue-specific migration pathways byphenotypically distinct subpopulations of memory T cells.Eur. J. Immunol. 22, 887.Mackay, C.R., Andrew, D.P., Briskin, M., Ringler, D.J., Butcher, E.C.,1996. Phenotype and migration properties of three major subsetsof tissue homing T cells in sheep. Eur. J. Immunol. 26, 2433.Smith, J.B., McIntosh, G.H., Morris, B., 1970. The traffic of cellsthrough tissues: a study of peripheral lymph of sheep. J. Anat.107, 87.264 A.G. Gossner et al. / Veterinary Immunology and Immunopathology 87 (2002) 261–264

Differential expression of homing molecules on recirculating lymphocytes from sheep gut, peripheral, and lung lymph.

Lymphocyte subset-speciﬁc and tissue-speciﬁc lymphocyte–endothelial cell recognition mechanisms independently direct the recirculation of lymphocytes from blood to lymph in sheep.

Lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes.

Naive and memory T cells show distinct pathways of lymphocyte recirculation.

Phenotype and migration properties of three major subsets of tissue homing T cells in sheep.

The origin of the cells in the efferent lymph from a single lymph node.

The output of cells in lymph from the popliteal node of sheep.

The trafﬁc of cells through tissues: a study of peripheral lymph of sheep.

Tissue-speciﬁc migration pathways by phenotypically distinct subpopulations of memory T cells.

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo.

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo.

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