β-amyloid-42 (Aβ42) and β-amyloid-40 (Aβ40), major components of senile plaque deposits in Alzheimer’s disease (AD), are considered neurotoxic and pro-inflammatory. In multiple sclerosis (MS), Aβ42 is upregulated in brain lesions and damaged axons. Here we found, unexpectedly, that treatment with either Aβ42 or Aβ40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. Aβ42 and Aβ40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive Th1 or Th17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo Aβ42 and Aβ40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major pro-inflammatory cytokines and chemokines were reduced in the blood following Aβ peptide treatment. Protection conferred by Aβ treatment did not require its delivery to the brain: adoptive transfer with lymphocytes from donors treated with Aβ42 attenuated EAE in WT recipient mice and Aβ deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with Aβ-treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of Aβ there is exacerbated clinical EAE disease progression. Since Aβ42 and Aβ40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.
Celastrol, an active compound extracted from the root of the Chinese medicine “Thunder of God Vine” (Tripterygium wilfordii), exhibits anticancer, antioxidant and anti-inflammatory activities, and interest in the therapeutic potential of celastrol is increasing. However, described side effects following treatment are significant and require investigation prior to initiating clinical trials. Here, we investigated the effects of celastrol on the adult murine hematopoietic system.
Animals were treated daily with celastrol over a four-day period and peripheral blood, bone marrow, spleen, and peritoneal cavity were harvested for cell phenotyping. Treated mice showed specific impairment of the development of B cells and erythrocytes in all tested organs. In bone marrow, these alterations were accompanied by decreases in populations of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP).
These results indicate that celastrol acts through regulators of adult hematopoiesis and could be used as a modulator of the hematopoietic system. These observations provide valuable information for further assessment prior to clinical trials.
Rationale: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. Methods: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. Results: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. Conclusions: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.
Anaphylaxis; Atopic; CD203c; Cockroach allergen; Flow cytometry; Human basophils; Omalizumab; Peanut allergy; Tree nut allergy
The ability of flow cytometry to allow fast single cell interrogation of a large number of cells has
made this technology ubiquitous and indispensable in the clinical and laboratory setting. A current limit to the potential of this technology is the lack of automated tools for analyzing the resulting data. We describe methodology and software to automatically identify cell populations in flow cytometry data. Our approach advances the paradigm of manually gating sequential two-dimensional projections of the data to a procedure that automatically produces gates based on statistical theory. Our approach is nonparametric and can reproduce nonconvex subpopulations that are known to occur in flow cytometry samples, but which cannot be produced with current parametric model-based approaches. We illustrate the methodology with a sample of mouse spleen and peritoneal cavity cells.
In this presentation, we outline ways in which current users of Fluorescence Activated Cell Sorting (FACS) can get more from their FACS work without undue effort. FACS technology development, and the emergence of new software support for various aspects of this technology are now cooperating in this effort.
The demonstration that CD4 T cell counts can be used to monitor HIV disease progression opened the way to the first clinical FACS application; the demonstration that stem cells can be sorted and transferred to appropriately pre-treated recipients now opens the way to new and constructive FACS uses in the future.
We hope that our readers join us in helping to achieve the goal of seeing more and better FACS data in the future.
Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in ∼5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.
B lymphopoiesis; V(D)J recombination; lineage restriction; hematopoiesis; stem cell; transcription
Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell–derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.
T cell recruitment; complement C5 and C5a; skin immunity; IgM response; T and B cell interactions
CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression.
We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA). This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid.
We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y) and demonstrate the respective roles of the each region in the regulation of CD5 transcription.
Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.
We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell–derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus clearance and survival rates compared with wild-type controls. Irradiation chimeras in which only either B-1 or B-2 cells lack the ability to secrete IgM show mortality rates similar to those of mice in which neither B-1 nor B-2 cells secrete IgM. Dependence on both sources of IgM for survival is partially explained by findings in allotype chimeras that broadly cross-reactive B-1 cell–derived natural IgM is present before infection, whereas virus strain–specific, B-2 cell–derived IgM appears only after infection. Furthermore, lack of IgM secreted from one or both sources significantly impairs the antiviral IgG response. Reconstitution of chimeras lacking B-1 cell–derived IgM only with IgM-containing serum from noninfected mice improved both survival rates and serum levels of virus-specific IgG. Thus, virus-induced IgM must be secreted in the presence of natural IgM for efficient induction of specific IgG and for immune protection, identifying B-1 and B-2 cell–derived IgM antibodies as nonredundant components of the antiviral response.
B cells; immunoglobulin M; immune protection; CD5+ B cell; respiratory tract
Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1–infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.
chemotaxis; thioredoxin; HTLV-1; migration
Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)–induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1β converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4+ and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)– induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.
Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline
Cα mRNA and mature Cα mRNA transcripts. Germline Cα and mature Cα transcripts were readily
detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-l-cell population in CD5+ B-1a cells and CD5- B-1b cells, it was shown that in vivo expression of germline
Cα and mature Cα transcripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.
B-1 cells; germline transcripts; IgA
Thymus-derived cells (T cells) that actively suppress production of IgG2a immunoglobulins carrying the Ig-1b allotype have been found in adult (SJL x BALB/c)F1 mice exposed to anti-Ig-1b early in life. The suppression is specific for Ig-1b. The allelic product, Ig-1a, is unaffected. Spleen, lymph node, bone marrow, or thymus cells from suppressed mice suppress production of Ig-1b by syngeneic spleen cells from normal F1 mice. When a mixture of suppressed and normal cells is transferred into lethally irradiated BALB/c mice, there is a short burst of Ig-1b production after which Ig-1b levels in the recipient fall rapidly below detectability. Pretreatment of the cells from the suppressed mice with antiserum specific for T cells (anti-Thy-1b) plus complement before mixture destroys the suppressing activity. Similar results with suppressor cells were obtained in vitro using Mishell-Dutton cultures. Mixture of spleen cells from suppressed animals with sheep erythrocyte (SRBC)-primed syngeneic normal spleen before culture suppresses Ig-1b plaque-forming cell (PFC) formation while leaving Ig-1a PFC unaffected. Treatment of the suppressed spleen with anti-Thy-1b before transfer removes the suppressing activity.
The mechanism of chronic allotype suppression in (SJL x BALB/c)F1 mice has been investigated by means of cell transfer studies. These mice are phenotypically negative for serum Ig-1b, the paternal allotype determinant on γG2a immunoglubulin, as a result of perinatal exposure to maternal anti-Ig-1b. When spleen or bone marrow (B) cells from suppressed mice were injected into irradiated BALB/c "indicator" hosts, detectable levels of Ig-1b were demonstrated in the sera of a majority of the recipients early after transfer. These results indicate that Ig-1b-producing cells or their precursors are present in the lymphoid tissues of suppressed mice, even though they are not expressed. Within 5–7 wk, it was no longer possible to detect Ig-1b in the sera of these hosts, although cells producing another paternal allotype (Ig-4b) were shown to persist. Control BALB/c mice, injected with spleen and B cells from normal mice, continued to produce high levels of immunoglobulin carrying this allotype. The disappearance of serum, Ig-1b occurred most frequently in the recipients of suppressed spleen cells. Similar results were obtained using a mixture of spleen cells from normal and suppressed mice. Ig-1b production in the recipient mice ceased within a few weeks, even though the majority of cells in the mixture were obtained from normal (nonsuppressed) donors. The data are interpreted as evidence that chronic allotype suppression in mice is actively maintained by cells which are resident in the lymphoid tissues, splenic cells being the most effective. These cells are capable of proliferating in a new host and exerting their suppressive influence on Ig-1b-producing cells and/or their precursors.
Long-term (chronic) allotype suppression, previously reported only in rabbits, is shown here to occur in at least one strain combination of mice as well. Close to 50% of the offspring of SJL (Igb) males mated to BALB/c (Iga) females immunized against the paternal allotype were found to be suppressed for Ig-1b (γG2a) at 6 months of age. These mice are called "chronically" suppressed. The percentage of offspring in this strain combination suppressed for the paternal allotype at 8 wk of age is the same as that seen in an earlier strain combination tested, [(C57 x BALB/c)F1], in which all mice recover from suppression by 10–12 wk. After 8 wk, two distinct patterns of long-term (chronic) suppression emerge in (SJL x BALB/c)F1 mice: a small number of these mice never produce detectable amounts of Ig-1b throughout their lives, while the majority produce detectable Ig-1b sporadically, sometimes over a period of several weeks, the level of which eventually falls below detectability. Attempts to "cure" suppression by destroying the existent lymphoid population and forcing endogenous repopulation in chronically suppressed animals were unsuccessful. Furthermore, attempts to restore Ig-1b production by injection of cells from syngeneic Iga/Igb donors into irradiated, chronically suppressed recipients were also unsuccessful, although the same cell inocula, when injected into irradiated BALB/c (Iga/Iga) mice produced high levels of gamma globulin carrying the allotype. These results suggest that long-term allotype suppression resulting from perinatal exposure of offspring to specific anti-allotype antibody (anti-Ig-1b), is not due merely to an absence of Ig-1b-producing cells or their progenitors, but appears to be an active process, which dominates physiologically over normal production.
Progeny mice were confronted with maternal γ-globulin of a different allotype by either back-cross mating, intercross mating, or by foster nursing. In all cases, many mice subsequently produced alloantibodies directed against the incompatible maternal type of IgG2a-globulin. In one series of experiments, immunologic tolerance to the maternally derived γ-globulin was demonstrated to exist in the period before formation of spontaneous antibody. The state of tolerance was then lost, unless maintenance injections of foreign γ-globulin were given. These studies demonstrate in a natural situation that maternally derived foreign proteins can first induce a state of immunological tolerance which is followed, after disappearance of the antigen, by a state of immunity. As such, this parallels the experimental induction of tolerance to foreign proteins by neonatal injections.
In the mouse, antibody directed against an immunoglobulin allotype, Ig-1b, passed from mother to offspring or injected into neonates, suppresses synthesis of immunoglobulin carrying Ig-1b. In allotype homozygotes as well as heterozygotes the allotype suppression is manifested both by a delay of several weeks in attaining initial detectable allotype levels and a reduction in allotype level continuing into adulthood. A possible mechanism for the differentiation of the immune system consistent with both the kinetics of suppression reported here for the mouse and the comparatively longer lived and more complete allotype suppression described for the rabbit is discussed. Evidence for a strong intralitter (as opposed to interlitter) correlation of age of onset of immunoglobulin allotype synthesis is presented.
Eight antigens of 7S γ2-immunoglobulins controlled by alleles at a single locus Ig-1, have been identified in mice. This locus has previously been shown to determine antigenic specificities on the F fragments of 7S γ2a-globulins. The reactions of these antigens with various isoantisera have shown that the antigens all cross react with one another. New methods for the analysis of antigenic specificities of soluble proteins are presented in detail. A sensitive method for detecting in the order of 0.01 µg of these isoantigens has been developed, based on the quantitative inhibition of precipitation of I125-labeled antigen. Cross-reactions of the antigens were analysed in inhibition assays and the data is compatible with the existence of a minimum of eight antigenic specificities. Each of the antigens is composed of different combinations of these specificities, with only one antigen having a specificity not present in any other. Sixty-eight mouse strains have been tested with specific isoantisera, and on the basis of the results, have been placed into the eight allele groups. Evidence for close genetic linkage of the Ig-1 locus and 11 chromosome markers has been sought and not found.