Analysis of monoclonal antibodies that recognize gamma delta T/null cells.

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine gamma delta TcR established the majority of null cells are gamma delta T cells. Use of this mAb in further comparisons demonstrated the gamma delta T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2- gamma delta T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.

Pig lymphocytes utilise mouse MAdCAM-1 to enter fetal gut xenografts in SCID mice.

Ileocecal junction (ICJ) and proximal intestine (PI) fragments from CD45(323-) allovariant fetal pigs were grafted subcutaneously into SCID mice. The xenografts were examined 8-12 weeks later using two-color immunohistology and the ICJ, but not PI, xenografts were found to contain three types of vessels. The first (the majority) was lined with mouse endothelium (mAb 9F1+), the second was lined with pig endothelium, and the third was chimeric. The ICJ vessels were specifically lined with mouse endothelium expressing MAdCAM-1, the mucosal addressin. Vessels lined with pig endothelium alone did not express the MAdCAM-1 epitopes. Radiolabeled allovariant pig peripheral blood lymphocytes (PBL) were introduced i.v. into the xenografted SCID mice, and entry into xenografts studied. Pig PBL were occasionally seen in MECA-367+ vessel walls after 4 h and within the ICJ but not PI xenografts after 24 h. This entry was specifically blocked by coinjection of the anti-MAdCAM-1 mAb MECA-367. The results demonstrate reendothelialization of xenografts by host endothelium that expresses its own addressin and is functional for xenogenic PBL.

The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs.

We have studied the role of E-selectin in leukocyte accumulation into Ag-specific cutaneous delayed-type hypersensitivity reactions in pigs sensitized to the topical application of 2,4-dinitro-1-fluorobenzene or to the intradermal injection of bacillus Calmette-Guérin. The delayed-type hypersensitivity reactions were shown to be specific for the sensitizing Ag and characterized by the up-regulation of E-selectin, as demonstrated by the uptake of tracer 99mTc-labeled monoclonal anti-E-selectin mAb and entry of 51Cr-labeled PBL and (111)In-labeled polymorphonuclear cells (PMN). Intravenous injection of 5 mg/kg of a F(ab')2 preparation of a monoclonal anti-E-selectin Ab at peak times of leukocyte entry resulted in a significant inhibition of entry of both PMN and lymphocytes. The anti-E-selectin Ab inhibited PMN recruitment by 70 to 90% and lymphocyte recruitment by 50 to 60%. In comparison, an anti-CD18 treatment reduced PMN recruitment by 70 to 90% and lymphocyte recruitment by 60 to 70% in this model. These data confirm an important role for E-selectin in the recruitment of both PMN and lymphocytes to sites of immune-based dermal inflammation.

Constitutive and inflammatory lymphocyte trafficking.

Here we provide a brief overview of lymphocyte trafficking with particular emphasis on the current state of knowledge in the pig. We discuss how the emphasis of research has changed since early studies in the 1960s and outline the current hypothesis of a multistep cascade for lymphocyte migration through specialized endothelia. During the last several years our research has focused mainly on lymphocyte migration in vivo. The inbred Babraham herd of MHC homozygous Large White pigs has allowed study of entry of either labelled (FITC or 51Cr) or unlabelled CD45 allotype-different donor lymphocytes and their subsets into various lymphoid, non-lymphoid and inflammatory tissues. The findings are considered under three different categories. Firstly, constitutive lymphocyte entry via 'high endothelial venules' (HEV-mediated), secondly, non-HEV-mediated lymphocyte homing and thirdly, lymphocyte entry into several models of inflammation with particular reference to the role of E-selectin. These findings demonstrate and underline the complexity and heterogeneity of lymphocyte homing, both at the whole population and subset level and yet, whilst a major step forward, the current hypotheses are perhaps too simple to explain much of this heterogeneity.

Lymphocyte subsets and adhesion molecules in cutaneous inflammation induced by inflammatory agonists: correlation between E-selectin and gamma delta TcR+ lymphocytes.

Kinetic and phenotypic heterogeneity in leukocyte subsets and adhesion-molecule expression is characteristic of many inflammatory conditions. We have studied the effect of various cytokines and inflammatory agonists on the type of leukocyte present and the adhesion molecules expressed in acute lesions (up to 3 days old) in porcine skin by immunohistology. Four major histocompatability complex-homozygous inbred pigs received replicate intradermal injections of IL-1 alpha, TNF-alpha, PMA, or PHA. Injections were timed so that lesions were obtained at 2, 4, 9, and 24 hours. Another two animals were studied at time points up to 72 hours. Leukocyte subsets and endothelial adhesion molecules were visualized on cryosections by use of monoclonal antibodies and alkaline phosphatase immunohistologic techniques. Substantial heterogeneity in leukocyte phenotypes was observed with all agonists, with lymphocyte subsets showing the greatest variation. Thus, CD2+ and gamma delta TcR+ (Null) T lymphocytes were present in all lesions, but to a varying extent such that few T cells were seen after PMA injection, approximately equal proportions of each after TNF-alpha, but substantially more gamma delta TcR+ (Null) lymphocytes were noted after PHA administration. Endothelial adhesion molecules were also variously affected, with E-selectin (CD62E) being transiently up-regulated by IL-1 alpha but CD62E showed early and sustained expression after TNF-alpha and PHA administration. The E-selectin ligand was demonstrated on infiltrating gamma delta TcR+ lymphocytes by use of a recombinant porcine E-selectin. The L-selectin ligand, identified by the mAb MECA-79, was only observed in late acute sites (< 24 hours) of TNF-alpha and PHA. Endothelial expression of class II major histocompatability complex was also variously up-regulated by all agonists. The results underline the heterogeneity of leukocyte phenotypes and endothelial adhesion molecule expression in acute cutaneous lesions dependent upon the nature of the inflammatory agonist and indicate an association between endothelial E-selectin and the presence of a gamma delta TcR+ (Null) T lymphocyte subset.

In vivo E-selectin upregulation correlates early with infiltration of PMN, later with PBL entry: MAbs block both.

The endothelial molecule E-selectin binds most leukocyte subsets in vitro. Yet its role in regulating the very different kinetics of inflammatory infiltration of different leukocyte subsets in vivo is unclear. The kinetics of E-selectin upregulation and polymorphonuclear leukocyte (PMN) and blood lymphocyte (PBL) localization in inflammation induced by interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), phytohemagglutinin (PHA), and phorbol myristate acetate (PMA) were investigated in a well-established inbred pig trafficking model. They differed markedly both for these three labeled indicators of inflammation and in each of the four inflammatory processes. In each, E-selectin upregulation correlated with early PMN entry and later with PBL infiltration but was more protracted than both. The importance of E-selectin was confirmed by marked inhibition of PMN and PBL entry (up to > 60%) by F(ab')2 anti-E-selectin. Involvement of other molecules was illustrated by similar or greater inhibition with anti-CD18 F(ab')2. We conclude that, like CD18, E-selectin is necessary for most PMN and PBL infiltration but alone is insufficient, consistent with the involvement of several alternative multistep molecular mechanisms in this entry.

Transfer of T-cell-mediated, antigen-specific delayed type hypersensitivity reactions to naive recipient inbred pigs.

Using inbred major histocompatibility complex-homozygous SLAb/b pigs, delayed-type hypersensitivity (DTH) reactions against either intradermal tuberculin (PPD) or topical 2,4-dinitro-1-fluorobenzene (DNFB) were transferred specifically by the intravenous injection of approximately 6 x 10(8) blood lymphocytes kg-1 bodyweight from donors sensitised, respectively, either with BCG or with DNFB into three-week-old piglets from an inbred litter. This antigen-specific, passively acquired sensitivity was revealed by three measures of DTH reactivity: first, macroscopic inflammation, which developed at the rate and intensity expected for actively acquired sensitivity to DNFB or PPD in older pigs; secondly, similarly enhanced local specific uptake of intravenously injected 51Cr-labelled normal lymphocytes (more than 35-fold for each); and, thirdly, histological evidence of markedly increased local infiltration of CD45+ lymphocytes and polymorphs, endothelial activation and the expression of adhesion molecules.

Genetically determined CD45 variant of value in leucocyte tracing in vivo in the pig.

This paper describes a monoclonal antibody (mAb), anti-pig CD45 (MAC323), that is directed against a polymorphic determinant. A monomorphic anti-pig CD45 mAb (K252.1E4) bound strongly to leucocytes from both MAC323+ and MAC323- pigs, demonstrating the absence of the epitope rather than the CD45 molecule. The MAC323 determinant was present on all leucocytes in positive pigs, exhibiting different expression levels on subsets (monocytes > lymphocytes > polymorphs), but was absent on red blood cells. Pigs lacking this determinant were healthy and grew normally. Careful selection of male and female SLAb/b pigs produced families that were either positive or negative for this epitope. Interbreeding within these families identified the genetic segregation of this variation, which is consistent with the CD45(323) epitope being inherited as a simple dominant autosomal gene. The lack of this determinant in certain lines of inbred pigs has been used to study the homing, lifespan and tissue distribution of donor unlabelled MAC323+ leucocytes and their subsets (using single- and two-colour immunocytological techniques) in MAC323- recipients after either intravenous injection or exchange transfusion. These results, identifying trafficking areas and subsets in constitutive lymphoid and inflammatory tissues, obtained using this genetic marker, extend those obtained previously using fluorescein isothiocyanate (FITC)-labelled donor cells.

The behaviour of monoclonal antibodies in the First International Pig CD Workshop reacting with gamma delta/Null T lymphocytes in the blood of SLAb/b line pigs.

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with gamma delta Null T-lymphocytes, defined by the binding of mAb w020/141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to gamma delta Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059-w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067-w071, w080, w091-w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAb/b line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020/141), which identifies all gamma delta Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

Major long-term changes in gamma delta T-cell receptor-positive and CD2+ T-cell subsets after neonatal thymectomy in the pig: a longitudinal study lasting nearly 2 years.

Blood leucocyte subsets in neonatally (20-day-old) thymectomized (Tx) and sham-thymectomized (STx) pigs were analysed 13 times over nearly 2 years. Tx piglets showed a persistent selective leucopenia, due mainly to a approximately 95% reduction in gamma delta null T cells which fell, with a circulating half-life of approximately 2 weeks, to approximately 0.3 x 10(6)/ml. This residual population was extrathymic in origin since it increased numerically at least approximately eightfold as the Tx pigs grew. Changes in other subsets were complex and affected by antigenic experience associated with weaning and with a change of accommodation at approximately 4 months postoperation (p.o.). Most major populations were increased long-term after thymectomy, especially after 3 months p.o. [i.e. surface (s)IgM+ B cells and CD2+, CD8+, major histocompatibility complex (MHC) class II+, CD4+ and double-positive CD4+ CD8+ T-cell subsets]. However, during the first 3 months, thymectomy caused a significant delay in development of CD8high and CD4+ T cells and, after 4 months p.o., a continuing lack of CD4only (single-positive) T cells. Fortuitous environmental antigenic stimulation caused a major transient lymphocytosis, with counts increasing 1.2-fold in STx and 3.5-fold in Tx pigs. This was largely due to an increase in CD2+ CD8+ MHC class II+ T cells, particularly in Tx pigs. The small residual thymus-independent gamma delta null subset also increased, while gamma delta T cells in STx pigs actually decreased. Evolving changes in expression of CD45, CD45R, CD44, CD18 and very late antigen type-4 (VLA-4) also occurred following thymectomy. Thus, the most persistent long-term effect of thymectomy, other than the lack of gamma delta null T cells, was the markedly increased numbers of double-positive (CD4+ CD8low) T cells, most of which expressed MHC class II and higher levels of adhesion molecules.

Porcine E-selectin: cloning and functional characterization.

E-selectin, a member of the selectin family, is believed to play an important role in mediating the initial adhesive events between leucocytes and the endothelium during inflammation. A monoclonal antibody against human E-selectin was found to cross-react with the porcine equivalent, a glycoprotein of 92,000 MW. Isolation of the cDNA for porcine E-selectin showed that it was 71% homologous with human E-selectin but had two less short consensus repeats. The porcine adhesion molecule could also support the adhesion of both porcine and human neutrophils. Expression of E-selectin on interleukin-1 alpha (IL-1 alpha)- or tumour necrosis factor-alpha (TNF-alpha)-activated porcine aortic endothelial cells in culture was prolonged, persisting for up to 48 hr. Binding studies using a chimeric molecule consisting of the lectin domain of porcine E-selectin and the epidermal growth factor (EGF) domain of human E-selectin fused to the human IgG constant region, further characterized porcine E-selectin as recognizing mainly granulocytic leucocytes and a subpopulation of lymphocytes.

Differences in E-selectin expression and leucocyte infiltration induced by inflammatory agents in a novel subcutaneous sponge matrix model.

We have developed a novel subcutaneous sponge matrix model in major histocompatibility complex (MHC) homozygous SLAb/b inbred pigs to study lymphocyte-endothelial cell interactions during inflammation. Polyether sponges were implanted subcutaneously and left for 12 days before injection of proinflammatory agonists. Implanted sponges became highly vascularized and showed markedly increased uptake of i.v.-injected 51Cr-labelled lymphocytes 5 hr after injection of tumour necrosis factor-alpha (TNF-alpha) (3000 U) or phytohaemagglutinin (PHA) (37 micrograms). Lower levels of traffic were seen in sponges 5 hr after injection with interleukin-1 alpha (IL-1 alpha) (3000 U) and no significant traffic occurred in sponges injected with phorbol 12-myristate 13-acetate (PMA) (15 ng) at 5 hr or PHA at 24 hr (compared to sponges injected with medium alone). Electron microscopy of control sponges revealed low numbers of infiltrating leucocytes and relatively 'flat' endothelium. Many more infiltrating leucocytes were present in PHA-injected sponges. However, no ultrastructural evidence was seen of any significant difference between control and activated endothelium. Immunocytochemistry of frozen sections from sponges showed that E-selectin expression was up-regulated markedly by TNF-alpha and PHA at 5 hr, only moderately by IL-1 alpha at 5 hr, and not at all by PMA at 5 hr. By 24 hr in PHA-injected sponges E-selectin expression had fallen markedly from the level seen at 5 hr. Flow cytometric analysis of cellular infiltrates dispersed from sponges injected with TNF-alpha, PHA, IL-1 alpha or medium alone, revealed differences in lymphocyte subset populations. The infiltrate in sponges injected with TNF-alpha 5 hr before removal was dominated by high numbers of CD2+ lymphocytes, whereas the infiltrate induced by PHA showed relatively higher levels of CD2- CD4-CD8- gamma delta T-cell receptor+ (TCR+) T cells revealed by population-specific monoclonal antibodies (mAb). This model, which permits harvesting of leucocytes and endothelial cells for manipulation in vitro, will be useful for the study of leucocyte-endothelial cell interactions in subacute and chronic inflammation.

Report on the behaviour of monoclonal antibodies in the First International Pig CD Workshop identifying the Null cell families.

Clustering analyses were carried out on data from five independent laboratories testing 22 monoclonal antibodies (mAbs) reacting with CD2-sIg-lymphocytes on 14 pig blood and/or tissue lymphoid target cells using cytofluorometry. This was coupled with extensive further studies on blood lymphocytes from normal and thymectomised SLAb/b inbred pigs. These mAbs formed two groups: those mainly identifying the large blood-borne thymus-dependent Null T cells (N) and those reacting with tissue and a small number of blood-borne thymus-independent lymphocytes (N'). Based on their tissue cell reaction patterns, the 10 N' mAbs formed three main groups: N'1A and B; N'2A and B; and N'3. The 12N mAbs fell into four groups N4-N7; N6 was divided into subgroups A-D. One N' (032) and two N mAbs (010 and 063) were unclustered. Based on these data, swine workshop cluster numbers were designated to groups N5 (021, 022 and 059) as SWC4, N6 (061 and 117) as SWC5 and N7 (020 and 141) as SWC6, the latter exceptionally as a single antibody, MAC320, since it is the 'type' mAb identifying effectively all blood Null T lymphocytes. Future research and workshops will have to define with a wider range of techniques the relationships, molecular properties and functional roles of the several new, perhaps novel, antigens identified by this family of fascinating, as yet still poorly defined, mAbs.

Characterization of E-selectin expression in vivo with use of a radiolabeled monoclonal antibody.

We have studied endothelial luminal surface expression of E-selectin in vivo in the pig. Intravenous interleukin-1 (IL-1, 5-micrograms/kg bolus +/- 50-ng.kg-1 x min-1 infusion for 2 h) induced E-selectin expression in many organs, as shown by immunostaining and selective clearance of intravenous 111In- or 99mTc-labeled anti-E-selectin monoclonal antibody (MAb 1.2B6) compared with radiolabeled immunoglobulin G1 control. Specific clearance of MAb 1.2B6 commenced 30-45 min after intravenous IL-1. Skin sites injected with IL-1, tumor necrosis factor, phytohemagglutinin, or phorbol myristate acetate at various times (45 min-24 h) before exsanguination showed specific accumulation of MAb 1.2B6 when 99mTc-MAb 1.2B6 and 111In-control immunoglobulin G1 were injected intravenously 10 min before exsanguination. This was maximal in 2-h IL-1 and tumor necrosis factor lesions and after 9 h in phytohemagglutinin and phorbol myristate acetate lesions. This novel approach has allowed us to quantify changes in vascular luminal expression of E-selectin in models of inflammation involving systemic and localized endothelial cell activation and has considerable potential for analyzing these changes in relation to leukocyte traffic and other manifestations of inflammatory responses in vivo.

Active lymphocyte traffic induced in the periphery by cytokines and phytohemagglutinin: three different mechanisms?

Phytohemagglutinin (PHA) injection induces transient protease-sensitive traffic of lymphocytes in skin and other tissues in several species. Examination of the possible roles of cytokines in such reactions showed that recombinant bovine and human tumor necrosis factor (TNF)-alpha potently induce dose-dependent lymphocyte traffic in pig skin (and in other tissues including the draining lymph nodes) with early kinetics and a morphology of the inflammatory reaction similar to that of PHA (peaking 9-12 h). Recombinant human interleukin (IL)-1 alpha also induces dose-dependent lymphocyte traffic, but it peaks at 4 h. Entry of labeled lymphocytes into inflammatory sites induced by PHA, TNF-alpha and IL-1 alpha, but not into normal skin, is inhibited by approximately 80% by their pretreatment with trypsin, indicative of the induction of endothelial determinants recognized by protease-sensitive surface molecules on the lymphocytes. Even the minimal lymphocyte traffic induced by interferon-gamma and lipopolysaccharide was similarly protease sensitive. At the earliest stage (approximately 2 h) of significant induction of lymphocyte entry by TNF-alpha and IL-1 alpha the inductive signal for each appears easily saturated. Thus lymphocyte entry is little increased by increasing low cytokine doses over 100-fold: However, these reactions are additive, and this was used to confirm that they are distinct from each other and from PHA. A further distinction was revealed by the homing of lymphocytes pretreated with pertussis toxin: such lymphocytes were greater than 90% inhibited in their homing to tissues through constitutive high endothelial venules (HEV) and greater than 60% inhibited in homing to TNF-alpha and IL-1 alpha skin sites, but unaffected in homing to PHA skin sites (like most non-HEV-mediated traffic). Moreover, potent chicken anti-TNF-alpha, which prevented TNF-induced lymphocyte entry, did not affect PHA-induced traffic. Thus, these three agents which induce peripheral lymphocyte traffic appear to involve different mechanisms as shown by differences in (i) their kinetics; (ii) the effect of anti-TNF-alpha and (iii) the effect of pertussis toxin treatment of the lymphocytes and by the fact that their inductive mechanisms are additive in effect.

Homing of blood, splenic, and lung emigrant lymphoblasts: comparison with the behaviour of lymphocytes from these sources.

The distinctive homing patterns of [125I]deoxyuridine-labelled lymphoblasts and 51Cr-labelled lymphocytes from the three sites--splenic and pulmonary venous emigrants, and peripheral blood--were demonstrated by study of their entry into a large number of tissues. These included the major organs such as the lung, liver, bone marrow, spleen, skin, and muscle, and several lymph nodes (LNs), Peyer's patches (PPs), tonsils and thymus, portions throughout the gastrointestinal tract and immunologically-stimulated sites. Major differences in homing of blood blasts (up to 4-fold) were found in particular tissue types, such as the different LNs, three PP types, and the three tonsils, and their distribution was quite different from lymphocytes. While splenic blasts behaved like blood blasts, lung blasts showed even more distinctive homing, e.g. to tissues with particular blast uptake, including spleen, stomach, bladder, portal and stimulated LNs, and liver and lung. While blast recovery data showed that all types homed well to bone marrow (about 25%) and the gastrointestinal wall (about 15%), the roughly 50% found in blood, muscle, skin, lung, and liver showed some homing preferences: blood blasts tended to stay more in blood, splenic blasts to home to muscle, and lung blasts to liver and lung. These studies have demonstrated that physiologically-derived lymphoblasts are not predominantly mucosal-homing but distribute through several non-lymphoid organs and that, even considering only those in the blood compartments, they show differences in homing preference.

Direct cytotoxicity mediated by allogeneic and isogeneic pig lymphocytes modified with fluorescein isothiocyanate.

Cell mediated cytotoxicity (CMC) is restricted by the major histocompatibility complex (MHC) in some animal models but not in others. In order to determine if there was MHC restriction in a CMC experimental model, mononuclear cells from blood of two lines of pigs, (1/1) and (6/6) which are homogeneous to the MHC products (SLA-ABCD), were used. Cytotoxic (1/1) cells were induced in vitro and in vivo against (1/1) mononuclear cells modified with fluorescein isothiocyanate (FITC) and tested against (1/1)-FITC isogeneic and (6/6)-FITC allogeneic cells, without showing MHC restriction of the cytotoxicity. The cytolysis was not due to antibody and K cells because fluoresceinated ovalbumin did not inhibit the test. It was not due to the "altered self" reaction against its own MHC products that sometimes may be induced by allogeneic responses, because the (1/1) anti (6/6) cytotoxic cells did not kill haptenated cells (1/1)-FITC. According to the results there was no evidence of MHC restriction of the CMC response of haptenated cells from pigs of (1/1) haplotype.