Kinetics and distribution of antigen-specific IgE-secreting cells during the primary antibody response in the rat.

A new assay system is described for the enumeration of antigen-specific IgE immunoglobulin-secreting cells (ISC) based on an enzyme-linked immunoabsorbent assay. Using this technique to monitor the organ distribution of OVA-specific ISC after primary immunization of rats, approximately 12,000 specific IgE ISC were detected at the peak of the response in the draining lymph nodes compared with 117,000 IgG ISC; the splenic anti-OVA response was restricted to the IgM class. Using plates precoated with anti-rat IgE instead of antigen, total IgE ISC were enumerated in normal and helminth-parasitized rats. The assay system detected up to 5 X 10(5) IgE ISC in mesenteric lymph nodes from parasitized animals compared with less than 50 in controls.

Microglia induce CD4 T lymphocyte final effector function and death.

Microglia, a type of tissue macrophage, are the only cells in the central nervous system (CNS) parenchyma to express some major histocompatibility complex (MHC) class II constitutively or to upregulate expression readily. They are thought to play a role in CD4 T cell activation in autoimmune diseases such as multiple sclerosis, as well as in neurodegenerative conditions, Alzheimer's disease in particular. We show here that highly purified MHC class II+ microglia when tested directly ex vivo do indeed support an effector response by an encephalitogenic myelin basic protein-reactive CD4 T cell line from which production of the proinflammatory cytokines, interferon gamma and tumor necrosis factor, is elicited, but not interleukin (IL)-2 secretion or proliferation. After this interaction, the T cells die by apoptosis. Other nonmicroglial but CNS-associated macrophages isolated in parallel stimulate full T cell activation, including IL-2 production, proliferation, and support T cell survival. Neither CNS-derived population expresses B7.1/B7.2. Resident macrophages that terminate effector T cells in tissues constitute a novel and broadly applicable regulatory measure of particular relevance to processes of self-tolerance against sequestered antigens.

Major histocompatibility complex-expressing nonhematopoietic astroglial cells prime only CD8+ T lymphocytes: astroglial cells as perpetuators but not initiators of CD4+ T cell responses in the central nervous system.

The potential of cells within the central nervous system (CNS) to initiate T lymphocyte responses is not known and was the subject of this study. Using the ability of virgin T lymphocytes to proliferate in a primary response to allogeneic determinants on antigen-presenting cells (APC), we have examined the capacity of major histocompatibility complex (MHC)-expressing astroglial cells to act as stimulators of primary and secondary T cell responses. Neither freshly isolated astrocytes nor primary astrocyte cultures pretreated with interferon gamma (IFN-gamma) to upregulate MHC class I and II expression stimulated unfractionated lymph node (LN) cell populations in the primary mixed lymphocyte reaction. In mixing experiments, astrocytes did not inhibit the T cell response to allogeneic LN stimulators. Purified responder CD4+ T cells also were not stimulated to proliferate or secrete interleukin 2 (IL-2) by MHC class I- and II-expressing astrocytes. In contrast to their inability to stimulate virgin, alloreactive CD4+ T cells, astrocytes were able to specifically stimulate an alloreactive CD4+ T cell line. Unprimed CD8+ T cells, however, exhibited some weak autonomous proliferation to astrocyte stimulators but this response was only substantial in the presence of exogenous IL-2, the latter predominantly being a CD4+ T cell product. Those CD8+ T cells responding in the presence of IL-2 were mainly T cell receptor alpha/beta+ IL-2 receptor (alpha chain)+, and a majority had shifted from high to low CD45R expression. Given the virtual dependence of CD8+ T cells in these studies, on CD4+ T cell help, and the complete absence of activation of this latter subset by astrocytes, it is clear that in the context of this resident CNS cell, further activation of either T cell subset by astrocytes within the CNS can only follow priming by another type of APC. The implications of these results for the induction of T cell responses in the CNS are discussed.

Resident macrophages (ramified microglia) of the adult brown Norway rat central nervous system are constitutively major histocompatibility complex class II positive.

A flow cytometric phenotype for isolated adult central nervous system (CNS) ramified microglia was previously defined (CD45low CD11b/c+) in the Lewis strain rat, that clearly distinguished these cells from all blood-derived leucocytes, the latter being CD45high. Consistent with the reported lack of major histocompatibility complex (MHC) expression in the CNS, isolated microglia were mostly MHC class II-. Employing these phenotypic criteria, we now show that a proportion of microglia in Brown Norway (BN) strain rats are constitutively MHC class II+. In spinal cord, up to 25% of microglia are distinctly positive and most have some level of expression. In situ staining of MHC class II+ microglial cells in BN rats indicates that positive cells are typical of ramified microglia on the grounds of both morphological appearance and anatomical location. In Lewis (LEW) rats, the few MHC class II-expressing cells isolated from the normal CNS are CD45high blood-derived cells and not resident microglia. After infection of both LEW and BN rats with a neurotropic murine hepatitis virus (MHV-JHM), MHC class II was rapidly upregulated on microglia in the BN but not in the LEW strain. In the latter, inflammatory cells were the predominant MHC class II-expressing population. Nevertheless, most microglia in the LEW strain could, after some delay, be induced to express MHC class II after transfer of an experimental autoimmune encephalomyelitis (EAE)-inducing encephalitogenic T cell line. Paradoxically, strains resistant to EAE (exemplified by the BN) contained more constitutive MHC class II-expressing microglia than susceptible ones, when a variety of strains were examined. The results clearly establish that the normal CNS may contain MHC class II-expressing cells that are a resident rather than a transient blood-derived population. It is significant that this expression is strain related, but there is no evidence that microglial cell constitutive MHC class II expression predisposes to EAE susceptibility.

An essential role for tumor necrosis factor in natural killer cell-mediated tumor rejection in the peritoneum.

Natural killer (NK) cells are thought to provide the first line of defence against tumors, particularly major histocompatibility complex (MHC) class I- variants. We have confirmed in C57BL/6 (B6) mice lacking perforin that peritoneal growth of MHC class I- RMA-S tumor cells in unprimed mice is controlled by perforin-dependent cytotoxicity mediated by CD3(-) NK1.1(+) cells. Furthermore, we demonstrate that B6 mice lacking tumor necrosis factor (TNF) are also significantly defective in their rejection of RMA-S, despite the fact that RMA-S is insensitive to TNF in vitro and that spleen NK cells from B6 and TNF-deficient mice are equally lytic towards RMA-S. NK cell recruitment into the peritoneum was abrogated in TNF-deficient mice challenged with RMA-S or RM-1, a B6 MHC class I- prostate carcinoma, compared with B6 or perforin-deficient mice. The reduced NK cell migration to the peritoneum of TNF-deficient mice correlated with the defective NK cell response to tumor in these mice. By contrast, a lack of TNF did not affect peptide-specific cytotoxic T lymphocyte-mediated rejection of tumor from the peritoneum of preimmunized mice. Overall, these data show that NK cells delivering perforin are the major effectors of class I- tumor rejection in the peritoneum, and that TNF is specifically critical for their recruitment to the peritoneum.

Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment.

Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] null mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.

Tumor necrosis factor (TNF)-dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35-55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF-/- mice. However, in the TNF-/- mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF-/- and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF-/- mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF-/- mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.

Tumor necrosis factor sustains the generalized lymphoproliferative disorder (gld) phenotype.

Tumor necrosis factor (TNF) and Fas ligand (FasL) play major roles in the homeostasis of the peripheral immune system. This becomes dramatically obvious in the absence of a functional FasL. Mice with such a deficiency develop a profound lymphadenopathy, splenomegaly, hypergammaglobulinemia, and strain-dependent systemic autoimmune disease, and succumb to premature death. It is consequently termed generalized lymphoproliferative disorder (gld). By contrast, TNF deficiency alone does not result in a striking phenotype. Thus, we sought to determine what role TNF might play in contributing to the gld phenotype by creating C57BL/6.gld.TNF(-/-) mice. Contrary to the expected outcome, mice deficient for both FasL and TNF had a substantially milder gld phenotype with regard to mortality, lymphoaccumulation, germinal center formation, and hypergammaglobulinemia. To confirm these data in a strain highly permissive for the phenotype, C3H/HeJ.gld and C3H.HeJ.lpr mice were treated with a TNF-specific monoclonal antibody. This transient neutralization of TNF also resulted in a significantly attenuated lymphoproliferative phenotype. We conclude that TNF is necessary for the full manifestation of the lymphoproliferative disorder, in particular playing a critical role in lymphoaccumulation. Most importantly, absence of TNF protects gld mice against premature death.

Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice.

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTalpha-/- mice, TNF-/- mice, and a new LTalpha-/- line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTalpha gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTalpha with soluble TNF receptor in unmanipulated wild-type or TNF-/- mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.

Generation of splenic follicular structure and B cell movement in tumor necrosis factor-deficient mice.

Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin alpha (LTalpha). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF-/- and TNF/LTalpha-/- mice. By creating radiation bone marrow chimeras from wild-type and TNF-/- mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor-IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF-/- recipients, but not into TNF/LTalpha-/- recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTalpha-/- mice because TNF/LTalpha-/- B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.

Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen.

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) alpha/beta lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTalpha- and LTbeta-deficient mice. Treatment of adult mice with antagonists of LTalpha1beta2 also leads to decreased BLC expression. These findings indicate that LTalpha1beta2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTalpha-, and LTbeta-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus-induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTalpha1beta2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen.

Down-regulation of the macrophage lineage through interaction with OX2 (CD200).

OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.

Induction of intratumoral tumor necrosis factor (TNF) synthesis and hemorrhagic necrosis by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in TNF knockout mice.

5,6-Dimethylxanthenon-4-acetic acid (DMXAA) is a new antitumor drug currently undergoing clinical trial. Administration of DMXAA to mice with tumors leads to cessation of tumor blood flow and the onset of tumor hemorrhagic necrosis, accompanied by the production of the cytokine tumor necrosis factor (TNF). Previous studies have shown that DMXAA induces both tumor and host cells to synthesize TNF and that induced intratumoral TNF production correlates with the antitumor activity of DMXAA. To explore the hypothesis that TNF production by tumor cells contributed to the induction of hemorrhagic necrosis by DMXAA, TNF-/- (C57Bl/6 background) mice were used as recipients for the s.c. implantation of (TNF positive) colon 38 adenocarcinoma. Tumors removed 24 h after treatment with DMXAA (66 or 100 micromol/kg) were found to be hemorrhagic and necrotic. Cells expressing TNF mRNA in tumors removed 2 h after treatment with DMXAA (160 micromol/kg) were found by in situ hybridization to be comparable in frequency and distribution with those in tumors from C57Bl/6 TNF-positive mice. However, the amount of TNF protein extracted from tumors from TNF knockout mice was lower than that from TNF-positive mice. Spleen and liver tissue from TNF knockout mice, in contrast to that from TNF-positive mice, produced no TNF mRNA. TNF protein was undetectable in liver and spleen tissue from TNF knockout mice, but was evident in tissue from TNF-positive mice. These results confirm that DMXAA has the novel ability of inducing tumors to synthesize TNF in situ.

Direct ex vivo flow cytometric analysis of human microglial cell CD4 expression: examination of central nervous system biopsy specimens from HIV-seropositive patients and patients with other neurological disease.

OBJECTIVE: To define a clear ex vivo flow cytometric phenotype for adult human microglia that would distinguish it from all other macrophage lineage cells in the central nervous system (CNS) or blood, and to utilize this phenotype to examine the activation state and CD4 expression of microglia freshly derived from CNS tissue of HIV-positive patients and those with other neurological diseases. DESIGN: Fresh human CNS tissue from both HIV-uninfected and HIV-infected individuals was obtained by biopsy or resection, and cells isolated immediately, labelled for flow cytometry and analysed. METHODS: A Percoll density gradient isolation technique and phenotypic characteristics used for rodent microglia were applied and modified. RESULTS: Resident microglia could clearly be defined by the flow cytometric phenotype CD45low CD4- CD11b+ CD11chigh major histocompatibility complex (MHC) class II+ CD26- CD14-. Assuming normally low-level MHC class II expression in the healthy CNS, it was likely that MHC class II positivity reflected underlying pathology necessitating biopsy or resection and appeared to be a 'leaky' activation marker. Microglia activation was observed in specimens from only six (35%) out of 17 HIV-uninfected but all four (100%) HIV-infected patients, defined strictly as any level of upregulation of CD4 expression, to produce the phenotype CD45low/medium CD4low CD11b+ CD1.1Chigh MHC class II+/+2 CD26- CD14-. Where examined by immunohistology, CD68 was also upregulated in these cases. CONCLUSIONS: When activated in situ, microglia express low levels of CD4 and this is always seen in tissue from HIV-infected patients. Using the flow cytometric phenotype established here, microglia from HIV-infected tissue can now be isolated in pure form and studied directly ex vivo.

The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells. An overview.

A solid-phase immunoenzymatic technique has been developed which allows the ready detection and enumeration of total- and antigen-specific immunoglobulin-secreting cells (ISC). The procedure involves the addition of putative ISC to plastic wells pre-coated with specific antigen or antisera. During incubation, the product of a single cell is immobilized by the solid phase at the point of release providing an immunological 'finger print' of the ISC which is subsequently developed by the application of appropriate enzyme-anti-Ig conjugates and an enzyme substrate which yields an insoluble product after incubation. Blue spots or 'ELISA plaques' are thus produced and can be counted macroscopically. This technique has been employed in rat, mouse and human systems and in each case appears to be of equivalent or greater sensitivity to existing haemolytic plaque techniques. The assay is particularly suited to the enumeration of antigen-specific ISC in which the antigen is difficult to couple to red cells or where a high degree of discriminating power is necessary as is required for example in the enumeration of IgE-ISC.

A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells.

A sensitive immunoassay is described for the detection of idiotype- and isotype-specific antibody-secreting cells (ASC), based upon the well established principles of ELISA. Single cell suspensions containing ASC are incubated on solid phase to which specific antigen has been chemically conjugated. Antibody attaches to the latter within the immediate microenvironment of the ASC, producing localized zones of bound antibody which are subsequently developed as visual 'spots' in the ELISA. This assay system has sensitivity and specificity at least equivalent to haemolytic plaque assays.

Isolation of encephalitogenic CD4+ T cell clones in the rat. Cloning methodology and interferon-gamma secretion.

In this report we detail a procedure for the cloning of a rat encephalitogenic T cell line and show that the methods normally employed for other species may not always be applicable. The two important differences to be described are, (i) that in these experiments where the parent T cell lines were generated with thymocytes as presenting cells, splenocytes were not suitable as a source of antigen-presenting or stimulator cells and (ii) semipurified forms of IL-2, specifically that derived from EL4 lymphoma cells, resulted in a much reduced cloning frequency and rate of T cell growth compared with cruder mixtures such as that derived from mitogen-stimulated splenocytes. Functional studies with clones derived from a strongly encephalitogenic (experimental autoimmune encephalomyelitis (EAE)-inducing) T cell line revealed that the clones had a reduced capacity to mediate EAE in recipient rats but were otherwise comparable to the parent line in terms of surface phenotype and fine antigen specificity. In an attempt to begin to identify the type of CD4+ T cells that may induce EAE we tested the clones and lines for secreted interferon-gamma by a sensitive ELISA, and showed that all clones secreted high levels of this factor.

Allele-specific PCR analysis for detection of the gld Fas-ligand point mutation.

The discovery of a naturally occurring missense point mutation in the gene encoding Fas-ligand (FasL/CD95L) in generalized lymphoproliferative disease (gld) mice has lead to the characterization of FasL as an important mediator of apoptosis. Further analysis of FasL function can be facilitated by crossing the gld mutation onto other mouse-strains, for example those carrying mutations affecting other molecules involved in apoptosis, or disease-prone genetic backgrounds. The success of this is dependent on a quick and reliable screening method. Here we report an allele-specific PCR for detection of the gld mutation. This approach permits the screening of back-crossed F1 progeny within one day, using whole blood samples as a source of genomic DNA. The technique is fast, robust, easily learnt, and unambiguous.

Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.

Dispase was used to digest central nervous system (CNS) tissue for isolation of the fixed tissue macrophage population (CNS microglia) and other leucocytes present. An apparent reduction in expression of some leucocyte cell surface markers was investigated further by addition of CD45b allotype-marked post-activated CD4+ T cells of known phenotype to CNS tissue preparations derived from a CD45a-expressing rat strain, before enzymatic treatment. Assessment of these T cells for expression of a range of surface molecules after completion of the isolation procedure revealed almost complete loss of expression of CD4 and CD25 and reduced expression of a number of other surface antigens.

Gene knock-out technology: a methodological overview for the interested novice.

Gene targeting by homologous recombination is a powerful technique, generating mouse strains with defined mutations in their genome. These genetically modified, 'designer' animals allow us for the first time to ask simple questions about elaborate and complex biological systems. Dissecting the function of individual components of the immune system is a perfect application of this technology. Although the techniques involved in the generation of gene knock-out mice are increasingly well defined, to many immunologists the language and concepts are confusing. This review presents the essentials of the technology in a form digestible by the non-expert.