Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice.

The pre-B cell receptor consists of immunoglobulin (Ig) mu heavy chains and surrogate light chain, i.e., the VpreB and lambda5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1(-/-) VpreB2(-/-) mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2-containing receptor in this process.

Leucocyte entry and endothelial E-selectin expression following intradermal Propionibacterium acnes administration.

Immune responses in porcine skin to intradermal inoculation of heat-killed Propionibacterium acnes (HKPA), the major bacterial agent associated with human inflammatory acne, were studied. Pigs were chosen as experimental animals because their skin is similar in structure and composition to that of man and because the use of genetically inbred pigs enables leucocytes to be transferred between animals without eliciting rejection responses. Two pigs were sensitized intradermally with 10 mg of HKPA and were challenged 2 weeks later with doses ranging from 1-100 microg of HKPA in various intradermal sites on the ventral aspect of the abdomen. Four further pigs, previously sensitized with Bacillus Calmette-Guérin (BCG) but not HKPA, were challenged with purified protein derivative (PPD) of bovine tuberculin and HKPA. Entry of(51)Cr-labelled peripheral blood lymphocytes (PBLs) over 48 h was studied in all the challenge sites. Peak PBL entry occurred at 4 h, remaining sustained up to 24 h. There was a dose-dependent effect of HKPA on the level of PBL entry, which was antigen-specific, as few leucocytes were seen in PPD-challenge sites in HKPA-sensitized pigs or in HKPA-challenged sites in BCG-sensitized pigs. There was also a substantial influx of(111)Indium-labelled neutrophils into the lesions. Lymphocytes present were predominantly of the CD3(+)CD2(+)T-cell subset, although gammadelta TCR(+)cells were present also, particularly after 24 h. E-selectin was markedly upregulated on dermal endothelium in the P. acnes sites. The histological infiltration and kinetics were similar to those reported in human inflammatory acne.

DNA profiling reveals remarkably low genetic variability in a herd of SLA homozygous pigs.

Inbred strains of rodents have become indispensable for a wide range of biological studies. It has generally been accepted that genetic uniformity is unlikely to be achieved before 20 generations of brother x sister matings discouraging attempts to inbreed larger mammals. Nevertheless, pigs, homozygous for the swine MHC haplotype SLA b/b, have been inbred at the Babraham Institute for almost thirty years and used for immunological studies. Since the herd had not been studied at the DNA level, DNA profiling at multiple hypervariable loci was performed and surprisingly little genetic polymorphism and extremely high inter-individual resemblance were observed reminiscent of that observed in inbred strains of mice.

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens.

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.

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.