We have developed a rigorous graph-theoretical algorithm for quantifying the shape properties of mutational lineage trees. We show that information about the dynamics of hypermutation and antigen-driven clonal selection during the humoral immune response is contained in the shape of mutational lineage trees deduced from the responding clones. Age and tissue related differences in the selection process can be studied using this method. Thus, tree shape analysis can be used as a means of elucidating humoral immune response dynamics in various situations.
Extracellular nucleotides such as ATP and NAD can profoundly affect the functions of lymphocytes, macrophages, and other cells. We have recently shown that extracellular NAD induces rapid apoptosis in naive T cells by a mechanism involving the ADP-ribosylation of cell surface molecules. In the present paper, we describe that T cells of different developmental stages differ in their sensitivity to NAD-induced apoptosis. Thymocytes were less susceptible than peripheral lymph node T cells, and freshly activated cells were more resistant than resting cells. Sensitivity to NAD-induced apoptosis generally correlated with expression of the ADP-ribosyltransferase ART2.2, which is not expressed on thymocytes and shed from peripheral T cells upon activation. Our findings suggest that NAD-induced apoptosis does not play a role during thymic selection of T cells, but rather may play a role by preventing the activation of unwanted bystander T cells during an immune response, and thus may participate in the control of autoimmunity.
The cellular prion protein (PrPc) is a glycolipid-anchored cell surface protein that usually exhibits three glycosylation states. Its post-translationally modified isoform, PrPsc, is involved in the pathogenesis of various transmissible spongiform encephalopathies (TSEs). In bovine species, BSE infectivity appears to be restricted to the central nervous system; few or no detectable infectivity is found in lymphoid tissues in contrast to scrapie or variant CJD. Since expression of PrPc is a prerequisite for prion replication, we have investigated PrPc expression by bovine immune cells. Lymphocytes from blood and five different lymph organs were isolated from the same animal to assess intra- and interindividual variability of PrPc expression, considering six individuals. As shown by flow cytometry, this expression is absent or weak on granulocytes but is measurable on monocytes, B and T cells from blood and lymph organs. The activation of the bovine cells produces an upregulation of PrPc. The results of our in vitro study of PrPc biosynthesis are consistent with previous studies in other species. Interestingly, western blotting experiments showed only one form of the protein, the diglycosylated band. We propose that the glycosylation state could explain the lack of infectivity of the bovine immune cells.
The thymus-specific serine protease Prss16 is highly expressed by the epithelial cells in the thymic cortex. It has been suggested to play an important role in the positive selection of T cells through the antigen presention pathway of the cortical antigen presenting cells. Recently, the gene ecoding Prss16 has been linked to insulin dependent diabetes mellitus (IDDM) susceptibility independent of HLA-DR3 suggesting the Prss16 may be involved in the development of autoimmune disease. Due to the similarities of the gene structure and expression pattern between the human and mouse genes, we compared Prss16 between non-obese diabetic (NOD) and non-obese non-diabetic (NON) mice. Analysis of the Prss16 coding region failed to identify any differences in sequence. Northern analysis and semi-quantitative reverse transcriptase polymerase chain reaction showed that the mRNA was equal in size and abundance in the two strains. In situ hybridization showed similar patterns of staining. Therefore, our data suggests that there is no significant different in the gene structure, transcription level, and expression pattern of Prss16 gene between NOD and NON mice.
Bone marrow progenitors migrate to the thymus, where they proliferate and differentiate into immunologically competent T cells. In this report we show that mice transgenic for SV40 T and t antigens under the control of the L-pyruvate kinase promoter develop, in a first step, thymic hyperplasia of both thymocytes and epithelial cells. Morphological studies (histology, immunohistolabeling and electron microscopy) revealed modifications of the thymic microenvironment and gradual expansion of medullary epithelial cells in 1 month-old mice, taking over the cortical region. Then, a thymic carcinoma develops. Two-color labeling of frozen sections identified the transgene in medullary epithelial cells. Flow cytometry analysis demonstrated a marked increase in mature CD4+ and CD8+ thymocytes in adult mice (39±10×106 in transgenic mice and 12±5×106 in age-matched controls). Furthermore, thymocyte export was disturbed.
Here we show that marginal zone (MZ)-B cells in rats can already be detected in neonatal spleen from two days after birth. At this time point, morphologically distinct MZs are not present yet and the vast majority of B cells in spleen are located in a concentric area surrounding the T cell zones (PALS). Before MZs are obviously detectable in spleen (14 days after birth), MZ-B cells seem to be enriched at the outer zones of the concentric B cell areas. Similar to adult rats, neonatal MZ-B cells are intermediate-sized cells that express high levels of surface (s)IgM and HIS57 antigen, and low levels of sIgD and CD45R (HIS24). We show here, however, that in contrast to adult MZ-B cells, MZ-B cells (and also recirculating follicular (RF)-B cells) in neonatal rats express higher levels of CD90 (Thy-1). In adult rats, expression of CD90 on the B cell lineage is confined to immature B cells. We speculate that the expression of CD90 on neonatal MZ-B cells may have implications for their responsiveness to polysaccharide (T cell-independent type 2) antigens.
Germinal centers (GC) are an essential part of the humoral immune response. They develop a clear structure during maturation: Centroblasts and centrocytes are separated into two zones, the dark and the light zone. The mechanisms leading to this specific morphology as well as the reason for zone-depletion during a later phase of the GC reaction have not clearly been revealed in experiment. We discuss and weigh possible mechanisms of dark and light zone development in the framework of two mathematical models. In a comparative approach we formulate constraints on typical lymphocyte velocities in GCs which are characteristic for the different proposed mechanisms.
In recent years, population declines related to viral outbreaks in marine mammals have been associated with polluted coastal waters and high tissue concentrations of certain persistent, lipophilic contaminants. Such observations suggest a contributing role of contaminant-induced suppression of cell-mediated immunity leading to decreased host resistance. Here, we assessed the effects of the prototypic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (B[a]P), and two polychlorinated biphenyls (PCBs), CB-156 and CB-80, on the T-cell proliferative response to mitogen in harbor seal peripheral lymphocytes. Despite the variability associated with our samples from free-ranging harbor seals, we observed a clear suppressive effect of B[a]P (10 uM) exposure on T cell mitogenesis. Exposures to 10 uM CB-156 and CB-80, and 1.0 and 0.1 uM B[a]P, did not produce significant depression in lymphoproliferation. Exposure to the model PAH at 10 uM resulted in a 61% (range 34-97%) average reduction in lymphoproliferation. We were able to rule out a direct cytotoxic effect of B[a]P, indicating that observed effects were due to altered T cell function. Based on our in vitro results, we hypothesize that extensive accumulation of PAH by top-trophic-level marine mammals could alter T cell activation in vivo and impaired cell-mediated immunity against viral pathogens.
Tissue recruitment of dendritic cells (DCs) is essential for antigen presentation. When latex particulates were injected intravenously into rats, DC precursors were recruited to the liver. Propionibacterium acnes also induced the recruitment of definite mouse DC precursors. These DCs initially showed a selective binding to Kupffer cells. In the Kupffer cell-depleted rats, DCs could neither be recruited to the liver nor adhere to sinusoidal walls. Pretreatment with varied monosaccharides in vitro showed that sugar residues consisting of N-acetylgalactosamine were necessary for this binding. Mouse DC precursors had CC-chemokine receptor 1 and 5, while granulama tissues and rat Kupffer cells expressed the corresponding chemokine, macrophage inflammatory protein-1α. Recruited DC precursors phagocytosed latex or bacteria and some of them soon translocated to hepatic nodes and induced the immune response there. We conclude that after invasion of pathogens, Kupffer cells not only scavenge them but also recruit DCs/DC precursors via chemokine- and N-acetylgalactosamine-mediated interactions. The accelerated DC traffic and the presence of blood-lymph translocation would induce rapid and efficient immune responses and thus contribute to the local defense to antigens within liver tissues as well as systemic defense to blood-borne antigens.
The plague, which the Board of Health had feared might enter with
the German troops into the Milanese, had entered it indeed,
as is well known; and it is likewise well known, that it paused not
here, but invaded and ravaged a great part of Italy. (A. Manzoni,
The Bethrothed, 1826)
Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed.
The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl
(NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely
related VH genes associated with the expression of the λ1 light chain.We investigated the anti-NP
primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic
alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed
although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount
a primary anti-NP response dominated by λ1 positive antibodies but fail to produce high affinity
NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged
GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.
Allergy and autoimmunity result from dysregulation of the immune system. Until recently, it was generally accepted that the mechanisms that govern these disease processes are quite disparate; however, new discoveries suggest possible common pathogenetic effector pathways. This review illustrates the concomitant presentation of these conditions and the potential relationship or common mechanism in some cases, by looking at the key elements that regulate the immune response in both allergic and autoimmunite conditions: mast cells, antibodies, T cells, cytokines, and genetic determinants. The parallel appearance of allergic and autoimmune conditions in the some patients may reveal that such aberrations of the immune system have a common pathophysiologic mechanism. Mast cells, which play a key role in allergic reactions, and the wealth of inflammatory mediators they express, make it likely that they have profound effects on many autoimmune processes. Activation of protein kinases by inflammatory cytokines and environmental stresses may contribute to both allergic and autoimmune diseases. The presence of autoantibodies in some allergic conditions suggests an autoimmune basis for these conditions. Because of the central role T cells play in immune reactivity, the T-cell receptor (TCR) loci have long been considered important candidates for common disease susceptibility within the immune system such as asthma, atopy, and autoimmunity. Immunomodulation is the key to a successful treatment of allergic and autoimmune conditions.
The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igα/Igβ heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation.
Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory (–1.5 and –15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.
The immune response of the neonate is poor and is dependent on passive immunity provided by maternal Ig. However, here we show that exposure of the neonate to environmental antigens induces a germinal center (GC) reaction. In the peripheral blood of premature infants one finds IgG class switched B cells expressing a selected V-gene repertoire. These data suggest that restrictions in the repertoire rather than immaturity of the immune system is responsible for the poor immune responses of the neonate.
Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210 kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.
Although it is well-established that macrophages can occur in distinct activation states, the molecular characteristics of differentially activated macrophages, and particularly those of alternatively activated macrophages (aaMφ), are still poorly unraveled. Recently, we demonstrated that the expression of FIZZ1 and Ym is induced in aaMφ as compared with classically activated macrophages (caMφ), elicited in vitro or developed in vivo during infection with Trypanosoma brucei brucei. In the present study, we analyzed the expression of FIZZ1 and Ym in caMφ and aaMφ elicited during Trypanosoma congolense infection and show that the use of FIZZ1 and Ym for the identification of aaMφ is not limited to T. b. brucei infection and is independent of the organ sources from which macrophages are obtained. We also demonstrate that FIZZ1 can be used to discriminate between different populations of aaMφ. Furthermore, we studied the effects of various stimuli, and combinations thereof, on the expression of FIZZ1 and Ym in macrophages from different mouse strains and demonstrate that regulation of the expression of FIZZ1 and Ym in macrophages is not dependent on the mouse strain. Finally, we show that these genes can be used to monitor the macrophage activation status without the need to obtain pure macrophage populations.
Since negative selection in the thymus is incomplete, some self-reactive T cells are able to mature and seed the periphery. To study how these T cells interact following encounter with the self-protein they recognize in the periphery, we have developed an adoptive transfer system in which HEL-specific TCR transgenic CD4 T cells are transferred to mice expressing HEL protein in the pancreas under the control of the rat insulin promoter. Here we show that after adoptive transfer of HEL-specific T cells functional tolerance is maintained despite evidence that the T cells encounter and respond to pancreas-expressed antigen. Even the provision of an additional activation stimulus by peripheral immunization with HEL protein is insufficient to induce the T cells to cause autoimmune tissue injury. However, in the presence of blocking anti-CTLA-4-mAb, immunized adoptive transfer recipients rapidly developed diabetes. These data suggest that the CTLA-4 pathway regulates the pathogenicity of antigen-specific T cells following a peripheral activation stimulus.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17's structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations.
Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the immunological destruction of intralobular bile ducts and serum anti-mitochondrial antibodies (AMA). Based upon previous work of oral tolerance and autoimmunity, we hypothesized that feeding the mitochondrial autoantigens of PBC would alter the clinical course and the level of antimitochondrial antibodies. The bovine pyruvate dehydrogenase complex (PDC) was purified and 5 mg fed in gelatin capsules to 6 patients with early stage PBC for 6 months. Antimitochondrial antibodies and liver biochemistries were measured at every 3 months for 12 months. The clinical trial was completed for all patients except for 1 who showed deterioration of pre-existing skin rash during treatment, which disappeared within 2 weeks after treatment was discontinued. However, after 1 year, neither the titers of AMAs nor liver biochemistries were significantly changed by this treatment. This is the first trial to test the efficacy of oral tolerance induction in PBC. However, the data, which limited in scope, did not demonstrate efficacy and further highlights the difficulties in showing continuing evidence of tolerance induction in autoimmunity.
Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by intrahepatic bile duct destruction and the production of anti-mitochondrial antibodies (AMA). The absence of an animal model has been a striking impedance in defining the molecular basis of disease. Previous work has suggested that SJL/J mice immunize with the pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen of PBC, leads to the development of lymphoid cell infiltration in portal tracts and a model system coined autoimmune cholangitis. We hypothesized that this pathology would be augmented if immunization occurred in the presence of IFN-γ injections. Accordingly, SJL/J mice were immunized with PDC-E2 and, for purpose of control, α-casein. Subgroups of mice were also treated with exogenous IFN-γ. As expected, mice immunized with PDC-E2, with or without IFN-γ, developed high titer AMAs. In contrast, mice immunized with α-casein, develop antinuclear antibodies. More importantly, the livers from mice immunized with PDC-E2 and/or those immunized with α-casein all displayed lymphoid cell infiltration to the portal tracts, irrespective of bile duct size. Indeed, there was no significant difference between the experimental and the control groups by histologic analysis. Thus, autoimmune cholangitis in these mice is antigen non-specific.
In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.
Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.
We have identified a novel mAb, SG31, which recognizes the mouse integrin α4 subunit. Unlike the epitopes recognized by other anti-α4 antibodies, the SG31 epitope is expressed on subpopulations of thymocytes and peripheral T cells. After manganese ion, but not phorbol myristic acetate activation, the epitope is induced and expressed on the majority of peripheral T cells. These data suggest that the SG31 epitope is an activation epitope and that manganese ions activate α4 integrins by inducing a conformational change. Comparative flow cytometric analyses showed that the SG31 epitope as well as the epitope detected by other anti-α4 antibodies is expressed on all B lineage cells. In the T lineage, expression of the α4 integrins is down-regulated during thymocyte development. Although mature thymocytes still express the α4 integrins, they lose almost entirely the activation epitope recognized by SG31. In contrast, the most immature thymocytes express high levels of this epitope. In the periphery, SG31 epitope is expressed mostly by activated T cells, in contrast to the overall population of T cells that express the α4 integrins at homogenous levels. These results suggest that the activation of the α4 integrins is parallel to that of T cells.
New Zealand Black (NZB) mice are a well-known animal model of human autoimmune disease. Although the mechanism for development of autoimmunity is unclear, NZB mice are well known for severe thymic microarchitecture abnormalities. It is thought that thymic dendritic cells (DC) may play a role in thymic education and contribute to the autoimmune process. To address this issue and, in particular, that qualitative and/or quantitative differences exist in thymic DC, we took advantage of a novel restriction analysis system that allow definition of differences in the expression of tyrosine kinases using highly enriched populations of thymic DC from NZB compared to BALB/c and C57BL/6 mice. The method chosen, restriction analysis of gene expression, allowed the determination of protein tyrosine kinase transcription profiles. We report herein that NZB mice have a significant upregulation of C-met compared to the control strains. The abnormality of the C-met transcription was confined to thymic DC. We believe that its abnormal expression reflects the resistance of thymic cells to apoptosis, which will ultimately lead to defects and/or abnormal signaling by the interaction of thymic DC and thymocytes. Further studies involving such interactions are under way.