Autoreactive B-1 B cells: constraints on natural autoantibody B cell antigen receptors

B-1 B-cells constitute a distinctive population of cells that are enriched for self-reactive B cell receptors (BCRs). These BCRs are encoded by a restricted set of heavy and light chains, including heavy chains that lack nontemplated nucleotide additions at the V-D and D-J joining regions. One prototype natural autoantibody produced by B-1 B cells binds to a cryptic determinant exposed on senescent red blood cells that includes the phosphatidylcholine (PtC) moiety. The V(H)11Vkappa9 BCR, which accounts for a large fraction of the anti-PtC specificity, is underrepresented in other B-cell populations, including newly formed B cells in bone marrow, and the transitional B cells, follicular B cells, and marginal zone B cells in spleen. Previous work has shown that V(H)11 heavy chains pair ineffectively with surrogate light chain (SLC) and so do not promote development in bone marrow, but instead allow fetal liver maturation because of a fetal preference for weaker pre-BCR signaling. Such inefficient SLC pairing constitutes one constraint on the maturation of B cells containing V(H)11 rearrangements that biases their generation to fetal development. Here, we examine another possible bottleneck to the B1 cell repertoire: light chain pairing with V(H)11 heavy chain, finding very significant preferences.     

The skewed heavy-chain repertoire in peritoneal B-1 cells is predetermined by the selection via pre-B cell receptor during B cell ontogeny in the fetal liver

As many as 5–15% of B-1 cells in the peritoneal cavityof adult mice produce antibodies reactive to phosphatidylcholine(PtC) and the vast majority of them express B cell receptors(BCRs) composed of V_H11-µH chains utilizing the J_H1 segmentand V9-L chains. This extremely skewed repertoire of PtC-reactiveB-1 cells is traditionally attributed to the expansion of particularclones in response to self or exogenous antigens. Here, we showthat the strong bias toward the J_H1 usage among V_H11-µHchains is already established prior to the BCR assembly, namelyat the transition from the large to the small pre-B cell stageduring B cell ontogeny in the fetal liver. Among V_H11-µHclones isolated from large pre-B cells where the J_H1 skewingwas not established yet, the J_H1 users showed the highest abilityto form pre-B cell receptor (pre-BCR) and to induce cellularproliferation and differentiation when expressed in fetal liverpro-B cells. Thus, the J_H1 users were positively selected andamplified at the pre-BCR checkpoint. When co-expressed withV9-L chains to form BCR, the J_H1 users almost exclusively conferredthe PtC reactivity on BCR even though other J_H users could alsoform BCR on the cell surface. Therefore, the pre-BCR-mediatedpositive selection of the J_H1 users among V_H11-µH chainsappears to be beneficial to the efficient generation of ‘innate-type’PtC-reactive B cells during the fetal B cell development, evenbefore the self-renewal or the antigen-driven clonal expansionof B-1 cells takes place in the peritoneal cavity.     

V{beta}6-bearing T cells are involved in resistance to Trypanosoma cruzi infection in XID mice

BALB.xid mice, carrying an X-linked mutation leading to theabsence of CD5⁺ B cells, are highly resistant to Trypanosomacruzi Infection. These mice clear blood parasites In the acutephase of infection and do not develop the inflammatory Infiltrationcharacteristically observed in the chronic phase of susceptiblestrains of mice. We have shown that the resistance of BALB.xldIs dependent on the production of high levels of IFN-y. Natural(adoptive foster) or artificial (In vivo Injection of blockingantibodies) treatments of BALB.xld induced deletion of CD4⁺and CD8⁺ cells bearing V_ß6 TCR. The absence of V_ß6lymphocytes considerably reduced resistance to infection. Furthermore,in BALB.xld lacking this minor fraction of the T cell repertoire,almost 50% of the IFN-y production is lost. This indicates thatV_ß6-bearing T cells are either directly or Indirectlyinvolved in the production of IFN-y and, thus, important foran effective immune response during the acute phase of experimentalChagas' disease.     

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR V(beta)8 during experimental cerebral malaria.

To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the alphabeta or gammadelta lineage (TCRalphabeta(-/-) or TCRgammadelta(-/-)). TCRgammadelta(-/-) mice were susceptible to CM, whereas all TCRalphabeta(-/-) mice were resistant, suggesting that T cells of the alphabeta lineage are important in the genesis of CM. The repertoire of TCR V(beta) segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the V(beta)8.1, 2 segments in the peripheral blood lymphocytes. Most V(beta)8.1, 2(+) T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69(+), CD44(high) and CD62L(low) phenotype surface markers. The link between the increase in V(beta)8.1, 2(+) T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the V(beta)8.1 and V(beta)8.2 chains, and in mice rendered deficient in V(beta)8.1(+) T cells by a mouse mammary tumor virus superantigen.     

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice

Autoimmunity is known to increase in aging. A possible factorcould be an alteration in the T cell repertoire wIth advancingage. Antibodies to the variable region of the ß chainof the TCR activate T cells and can serve as probes for analysisof the T cell repertoire. We have used V_ß3 and V_ß17aantibodies to determine the presence and functionality of normallydeleted T cells bearing potentially self-reactive TCR in peripherallymphoid tissue and blood from aged (SJL/J x BALB/c) F₁ LAF₁and BALB/c mice. Although an occasional 20- to 24-month-oldmouse exhibited V_ß3⁺ or V_ß17a⁺ T cells intheir lymph nodes or peripheral blood lymphocytes (PBL) slightlyabove the range for normal young mice of these I-E⁺ strains,there was no striking ‘escape’ from the normal thymicdeletion process. However, responsiveness to anti-V_ß3and anti-V_ß17a was slightly higher In aged, and particularlyIn aged thymectomlzed (TX), than in young mice. This was incontrast to proliferative responses to stimulation with antibodyto the normally expressed V_ß8 which were lower inthe lymph nodes from aged than from young mice. The PBL of some30- to 36-month-old mice were also examined. Enhanced numbersof ‘forbidden’ V_ß bearing T cells wereseen more frequently at this age. In spite of the age-relateddecrease in overall CD4/CD8 T cell ratios in all organs, themice with relatively high V_ß17a⁺ T cells exhibitedproportionally more CD4⁺ cells in that V_ß population.We conclude that the ‘forbidden’ T cells that respondto anti-V_ß stimulation in the 20- to 24-month-oldmice are most likely of extra-thymic origin, since they weremore readily detectable in aged TX mice. Potentially self-reactiveCD4 (and CD8) single-positive T cells were detectable in PBLonly in very aged (30–36 months old) euthymic mice.     

Kinetics of peritoneal B-1a cells (CD5 B cells) in young adult mice

In the mouse, conventional B cells are continuously generated from precursor cells located in the bone marrow (BM), whereas the small subset of B-1 cells (formerly called Ly-1 B cells) constitute a self-replenishing population of cells. Here we studied the kinetics of murine peritoneal B-1a cells (i.e. B-1 cells expressing CD5). The actual number of B-1a cells in the peritoneal cavity that are proliferating, as detected by metaphase arrest and S-phase index, was below detection level, indicating that these cells do not divide significantly at this anatomical location. To establish the life-span of B-1a cells we used long-term administration of 5-bromo-2′-deoxyuridine in combination with three-color immunocytology on cytospin preparations. The renewal rate of peritoneal B-1a cells was 1.3% per day representing a 50% renewal time of 38 days. Splenic B cells and popliteal lymph node B cells (predominantly conventional B cells) showed an almost identical renewal rate of 1.1% per day. The data show that peripheral B cells from various lymphoid tissues and locations do not differ significantly in their renewal capacity, even though there are differences in their developmental origin.     

Neonatal tolerance to MIs-1a determinants: deletion or anergy of V beta 6+ T lymphocytes depending upon MHC compatibility of neonatally injected cells.

Recent investigations in mice revealed that natural immunological tolerance to endogenous minor lymphocyte-stimulating locus 1a (MIs-1a) antigen correlates primarily with deletion of MIs-1a-specific V beta 6+ T lymphocytes in the thymus. Similar mechanisms account for acquired tolerance in some instances since the neonatal injection of MIs-1a-expressing MHC compatible cells in neonatal mice within the first 24 h of life causes clonal deletion of V beta 6+ T cells. Here we demonstrate that V beta 6+ T cells are not deleted in mice neonatally treated with MIs-1a spleen cells expressing allogeneic H-2 molecules. However, when such non-deleted V beta 6+ T cells were tested in vitro, no interleukin 2 (IL-2) secretion or proliferation was observed after MIs-1a stimulation. This non-responsive state could be overcome by addition of exogenous IL-2, consistent with the fact that V beta 6+ cells enlarged and expressed IL-2 receptors upon MIs-1a stimulation. Furthermore, the same neonatally treated mice showed in vitro functional unresponsiveness of cytotoxic T cells but not of IL-2-secreting cells specific for the tolerated allogeneic MHC antigens. Taken together, our data indicate that neonatal tolerance to MIs-1a can be accomplished by either clonal deletion or clonal anergy, and that it does not necessarily correlate with tolerance to MHC determinants.     

The role of alpha(5)beta(1)-integrin in the IGF-I-induced migration of extravillous trophoblast cells during the process of implantation

The role of integrins in the processes of adhesion and migration makes them attractive potential participants in the complex events of embryo implantation and placentation. Recently, the role of the alpha(v)beta(3)-integrin pathway was shown in the insulin-like growth factor-I (IGF-I)-stimulated migration of extravillous trophoblast (EVT) cells. This study was designed to investigate the role of alpha(5)beta(1)-integrin in this respect. Using cultured EVT cells, migration assays were carried out for IGF-I-treated or untreated cells in the presence or absence of the GRGDSP and GRGESP hexapeptides, alphaIR3, and a blocking antibody against alpha(5)beta(1)-integrin. Immuno-electron microscopy and immunofluorescent staining were performed to localize the distribution of alpha(5)beta(1)- and alpha(v)beta(3)-integrins, Rab5a, paxillin, phospho-FAK (pFAK), and vinculin. The results showed that IGF-I-induced migration of EVT cells was abolished following treatment with GRGDSP hexapeptide, alphaIR3, and a blocking antibody against alpha(5)beta(1)-integrin. Further, statistical analysis showed that the area-related numerical density of the alpha(5)beta(1)-integrin in the perinuclear regions was significantly higher than in the cell extensions. Immunocytochemical experiments demonstrated an up-regulation in internalization rate of alpha(5)beta(1)-integrin in IGF-I-stimulated EVT cells. Furthermore, alpha(5)beta(1)-integrin exhibited co-localization with Rab5a, but not with alpha(v)beta(3)-integrin, pFAK, paxillin, and vinculin at the focal adhesions of the EVT cells. Taken together, these findings suggest an essential role for alpha(5)beta(1)-integrin in IGF-I-promoted migration of EVT cells. It is possible therefore that IGF-I-induced internalization of alpha(5)beta(1)-integrin may be an important event during the migration of EVT cells in the complex processes of implantation and placentation.     

Thymus-derived cytokine(s) including interleukin-7 induce increase of T cell receptor α/β+ CD4−CD8− T cells which are extrathymically differentiated in athymic nude mice

Extrathymic T cell differentiation pathways have been reported, although the thymus is the main site of T cell differentiation. The thymus is also known to produce several cytokines that induce proliferation of thymocytes. In the present study, we investigated the influence of thymus-derived cytokines on extrathymic T cell differentiation by intraperitoneal implantation with a diffusion chamber which encloses fetal thymus (we named it fetal thymus-enclosed diffusion chamber, FTEDC) in athymic BALB/c nu/nu mice. Increase in number of T cells bearing T cell receptor (TcR) α/β was detected in lymph nodes and spleens of FTEDC-implanted nude mice 1 week after implantation, whereas no such increase was detected in control nude mice implanted with a diffusion chamber without thymus. The FTEDC-induced increase of T cells was suppressed by intraperitoneal injection of anti-interleukin-7 monoclonal antibody (mAb). The TcR α/β T cells in FTEDC-inplanted BALB/c nu/nu mice preferentially expressed Vβ11, although Vβ11-positive T cells are deleted in the thymus of euthymic BALB/c mice by clonal elimination of self-superantigen Dvb 11-specific T cells. TcR α/β T cells in FTEDC-implanted nude mice were of CD4^?CD8^? phenotype and showed no proliferative response against anti-TcR monoclonal antibody stimulation. These results suggest that the thymus can induce extrathymic T cell differentiation through the influence of thymus-derived cytokine(s) including interleukin-7, and that such extrathymically differentiated T cells have acquired only a little or no ability for proliferation when they recognize antigen by their TcR.     

The treatment of systemic lupus erythematosus (SLE) in NZB/W F1 hybrid mice; studies with recombinant murine DNase and with dexamethasone

The effects of recombinant mouse DNase on a well established murine model of spontaneous SLE have been evaluated. Daily intraperitoneal injections of DNase were given to female NZB/NZW F₁ mice during the period of disease development from 4 to 7 months of age or at the height of disease activity from the age of 7 months for 3 weeks. This treatment was compared with the injections of diluent and with an immunosuppressive dose of dexamethasone. The effects of treatment were evaluated using the immunological parameters of disease activity (antinucleoprotein antibody, immune complexes, serum immunoglobulins, anti-cardiolipin antibodies), proteinuria, serum creatinine and renal histopathology (light microscopy, immunofluorescence and electron microscopy). The dose of dexamethasone used (1 mg/kg per day from the age of 4 months) was sufficient to suppress the development of lupus entirely. Treatment with DNase starting at the age of 4 months postponed the development of the disease by about 1 month and extended the period from the onset of disease to death by about 30%. Mice treated for 3 weeks during the most active phase of the disease at 7 months of age showed more dramatic effects. Proteinuria and serum creatinine were significantly reduced and renal histopathology was strikingly less severe than in the control group. Immune complexes involving DNA-containing antigens are believed to play a crucial role in the pathogenesis of SLE. DNA-nucleoprotein, even in immune complexes, can be destroyed by DNase. This enzyme therefore provides a rational way to interfere with the disease process. The results reported here encourage a trial of recombinant human DNase in human SLE and lupus nephritis.     

High frequency of circulating gamma delta T cells with dominance of the v(delta)1 subset in a healthy population

TCR gamma delta(+) cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V(delta)1(+) cells constitute a minority of these cells. In contrast to TCR alpha beta(+) cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequencies of V(delta)1(+) cells are found in several diseases, their function remains obscure. Here we show that the frequency of peripheral blood gamma delta T cells in healthy West Africans is about twice that of Caucasians, mainly due to a 5-fold increase in V(delta)1(+) cells, which is consequently the dominant subset. No age dependency of V(delta)1 frequencies was identified and the V(delta)1(+) cells in the African donors did not show preferential V(gamma) chain usage. Analysis of the CDR3 region size did not reveal any particular skewing of the V(delta)1 repertoire, although oligoclonality was more pronounced in adults compared to children. The proportions of CD8(+), CD38(+) and CD45RA(hi)CD45RO(-) cells within the V(delta)1(+) subset were higher in the African than in the European donors, without obvious differences in expression of activation markers. No significant correlations between levels of V(delta)1(+) cells and environmental antigens or immunological parameters were identified. Taken together, the evidence argues against a CDR3-restricted, antigen-driven expansion of V(delta)1(+) cells in the African study population. Our study shows that high frequencies of TCR gamma delta(+) cells with dominance of the V(delta)1(+) subset can occur at the population level in healthy people, raising questions about the physiological role of V(delta)1(+) T cells in the function and regulation of the immune system.     

The neuroprotective role of tissue inhibitor of metalloproteinase-2 in MPP- or 6-OHDA-treated SK-N-BE(2)C and SH-SY5Y human neuroblastoma cells

Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases (MMPs), and the aberrant expressions of MMPs are strongly associated with neuroinflammation and neuronal cell death. In the present study, we found that two well-known dopaminergic neurotoxins, MPP⁺ and 6-OHDA, reduced TIMP-2 expression at the mRNA and protein levels in two human neuroblastoma cell lines (SK-N-BE(2)C and SH-SY5Y). To investigate the role of TIMP-2, these cells were transfected with TIMP-2 expression plasmid and viabilities were compared after treating cells with MPP⁺ or 6-OHDA. It was found that TIMP-2 overexpression attenuated the cell deaths induced by MPP⁺ or 6-OHDA, and that the degree of protection conferred was greater for MPP⁺-treated cells. Furthermore, the introduction of TIMP-2 siRNA into SK-N-BE(2)C cells aggravated the cell deaths induced by MPP⁺ or 6-OHDA. These findings collectively show that endogenously expressed TIMP-2 has a neuroprotective role, and they imply that the inhibition of TIMP-2 expression by MPP⁺ or 6-OHDA may contribute, in part, to neuronal cell death. These findings suggest that TIMP-2 expressional enhancement provides a potential therapeutic strategy for the treatment of neurodegenerative diseases such as Parkinson's disease.     

Ultrastructure of spinal anterior horn cells in human Niemann‐Pick type C (NPC) patient and mouse model of NPC with retroposon insertion in NPC1 genes

Niemann‐Pick disease type C (NPC) is a neurovisceral lipid‐storage disease. Although NPC patients show lipid storage in anterior horn cells of the spinal cord, little information is available regarding the electron microscopic analyses of the morphologies of intra‐endosomal lipid like‐materials in the anterior horn cells of NPC patients. In this study, we elucidated the intra‐endosomal ultrastructures in spinal anterior horn cells in an NPC patient, as well as in mutant BALB/c NPC1^?/? mice with a retroposon insertion in the NPC1 gene. These morphologies were classified into four types: vesicle, multiple concentric sphere (MCS), membrane, and rose flower. The percentages of the composition in the NPC patient and NPC1^?/? mice were: vesicle (55.5% and 14.9%), MCS (15.7% and 3.4%), membrane (23.6% and 57.1%), and rose flower (5.2% and 24.6%), respectively. Formation of the intra‐endosomal structures could proceed as follows: (i) a vesicle or MCS buds off the endosome into the lumen; (ii) when a vesicle breaks down, a membrane is formed; and (iii) after an MCS breaks down, a rose flower structure is formed. Our new finding in this study is that ultrastructural morphology is the same between the NPC patient and NPC1^?/? mice, although there are differences in the composition.     

Eating ourselves to death (and despair): The contribution of adiposity and inflammation to depression

Obesity and related metabolic conditions are of epidemic proportions in most of the world, affecting both adults and children. The accumulation of lipids in the body in the form of white adipose tissue in the abdomen is now known to activate innate immune mechanisms. Lipid accumulation causes adipocytes to directly secrete the cytokines interleukin (IL) 6 and tumor necrosis factor α (TNFα), but also monocyte chemoattractant protein 1 (MCP-1), which results in the accumulation of leukocytes in fat tissue. This sets up a chronic inflammatory state which is known to mediate the association between obesity and conditions such as cardiovascular disease, type 2 diabetes, and cancer. There is also a substantial literature linking inflammation with risk for depression. This includes the observations that: (1) people with inflammatory diseases such as multiple sclerosis, cardiovascular disease, and psoriasis have elevated rates of depression; (2) many people administered inflammatory cytokines such as interferon α develop depression that is indistinguishable from depression in non-medically ill populations; (3) a significant proportion of depressed persons show upregulation of inflammatory factors such as IL-6, C-reactive protein, and TNFα; (4) inflammatory cytokines can interact with virtually every pathophysiologic domain relevant to depression, including neurotransmitter metabolism, neuroendocrine function, and synaptic plasticity. While many factors may contribute to the association between inflammatory mediators and depression, we hypothesize that increased adiposity may be one causal pathway. Mediational analysis suggests a bi-directional association between adiposity and depression, with inflammation possibly playing an intermediary role.