bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of mouse CD40. Ligation of CD40 activates B cells via a Ca++‐dependent, FK506‐sensitive pathway
Authors: Klaus Gerry G. B. , Choi Michael S. K. , Holman Mary
Published: March 2007

The immunosuppressive drugs cyclosporine A (CsA) and FK506 selectively abrogate Ca++‐regulated activation pathways in both T cells and B cells. We show here that anti‐CD40‐induced murine B cell proliferation, in the presence or absence of interleukin‐4 (IL‐4) is, like the response to anti‐immunoglobulin (Ig), abrogated by 1–5 ng/ml (concentration causing 50% inhibition ca. 1 nM) FK506. However, the effects of the drug on proliferative responses elicited by anti‐Ig or anti‐CD40 differ significantly: firstly, the response to anti‐CD40 + IL‐4 becomes completely FK506‐resistant within 24 h, whilst that to anti‐Ig remains sensitive significantly longer. Secondly, stimulating B cells concurrently via CD40, surface Ig (sIg) and IL‐4 receptors invokes an FK506‐resistant activation pathway. We previously reported that ligation of either sIg or CD40 receptors, in conjunction with IL‐4, induces the transcription factor, nuclear factor of activated T cells (NF‐AT) in B cells, via a CsA/FK506‐sensitive pathway. However, NF‐AT induction elicited by anti‐Ig/anti‐CD40/IL‐4 is still FK506 sensitive, implying that the drug resistance of the response to these three stimuli involves additional components than NF‐AT. These results indicate that CD40 activates murine B cells via a Ca++‐dependent pathway. In agreement with this, anti‐CD40 induces a modest increase in intracellular Ca++ levels in these cells, which appears to be largely due to Ca++ influx through the plasma membrane.
Interleukin‐8 differentially modulates interleukin‐4‐ and interleukin‐2‐induced human B cell growth
Authors: Kimata Hajime , Lindley Ivan
Published: March 2007

The effect of interleukin‐8 (IL)‐8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL‐8 inhibited IL‐4‐induced growth of B cells costimulated with anti‐μ antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose‐dependent fashion. In contrast, IL‐8 did not inhibit IL‐2‐induced growth of B cells. The IL‐8‐mediated inhibition was specific, since it was blocked by anti‐IL‐8 mAb but not by control IgG1. Moreover, anti‐tumor necrosis factor‐α (anti‐TNF‐α) Ab blocked IL‐8‐mediated inhibition. On the other hand, TNF‐α, but not other cytokines including IL‐1β, IL‐3, IL‐5, IL‐6, interferon‐α (IFN‐α) or IFN‐γ, inhibited IL‐4‐mediated growth, and inhibition by TNF‐α was blocked by anti‐TNF‐α Ab but not by control IgG. IL‐4 had no effect on TNF‐α binding by B cells while it decreased TNF‐α production by B cells. IL‐8 had no effect in binding of IL‐4, IL‐2 or TNF‐α by B cells, however, it enhanced TNF‐α production by B cells. These results indicate that IL‐8 inhibited IL‐4‐induced human B cell growth by enhancement of endogenous TNF‐α production.
Interleukin‐12 production by human polymorphonuclear leukocytes
Authors: Cassatella Marco A. , Meda Lucia , Gasperini Sara , D'Andrea Annalisa , Ma Xiaojing , Trinchieri Giorgio
Published: March 2007

Human polymorphonuclear leukocytes (PMN) stimulated by lipopolysaccharide (LPS) produce interleukin‐12 (IL‐12). Both the free IL‐12 p40 chain and minute amounts of the biologically active IL‐12 p70 heterodimers are produced by PMN. Interferon‐γ (IFN‐γ) enhanced the LPS‐induced secretion of both the free IL‐12 p40 chain and the p70 heterodimer by approximately fivefold. As observed for other IL‐12‐producing cell types, the ratio of free p40 chain to p70 heterodimer secreted by LPS‐stimulated PMN was approximately 20:1. LPS induced a 100‐fold increase of IL‐12 p40 mRNA, but had minimal effect on p35 mRNA accumulation, IFN‐γ enhanced the LPS‐induced accumulation of p40 mRNA and directly induced a several‐fold increase in the accumulation of p35 mRNA. Therefore, the combined effect of LPS and IFN‐γ induced sufficient expression of both p40 and p35 to attain production of the biologically active p70 heterodimer at physiologically relevant concentrations. The ratio between p40 and p35 mRNA abundance in PMN stimulated with both LPS and IFN‐γ was approximately 200:1, explaining the secretion of the free p40 chain in much higher concentrations than the p70 heterodimer. IL‐10, an inhibitor of the production of various cytokines in PMN, also suppressed IL‐12 mRNA accumulation and secretion by PMN. Because of the important immunoregulatory function of IL‐12, in particular induction of IFN‐γ production and facilitation of T helper cell type 1 response, the ability of PMN to produce IL‐12 suggests that neutrophils may play an active role in the regulatory interaction between innate resistance and adaptive immunity.
Anti‐acetylcholinesterase antibodies display cholinesterase‐like activity
Authors: Johnson Glynis , Moore Samuel W.
Published: March 2007

A monoclonal antibody (mAb) raised against human acetylcholinesterase (AChE) was found to have catalytic activity. A similar phenomenon was observed in a polyclonal antibody raised against the same antigen. The antibodies were demonstrated to be pure, and no contamination with either AChE or butyryl‐cholinesterase was found. Both antibodies hydrolyzed acetylthiocholine, an AChE substrate, and the mAb followed Michaelis‐Menten kinetics. Six other mAb and one other polyclonal antibody showed no evidence of catalytic activity. This development of cholinesterase‐like behavior by certain anti‐AChE antibodies may have arisen by stable complexation of the enzyme with a substrate or inhibitor during antigen presentation. This phenomenon may have implications for the diagnostic measurement of AChE activity as well as in assessing the immunological reasons for the markedly raised AChE level in developmental conditions such as Hirschsprung's disease.
Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain
Authors: Busch Robert , Vturina Iryna Y. , Drexler Johannes , Momburg Frank , Hämmerling Günter J.
Published: March 2007

In normal antigen‐presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post‐Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP‐mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H‐2Ak MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact Ak αβ dimers of biosynthetically labeled Ii+ cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate‐(SDS) stable 55‐kDa peptide: Ak complexes were only found in the Ii+ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known Ak‐binding peptides bound stably to Ak molecules on Ii− cells, could be co‐purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of Ak: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.
Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin‐2
Authors: Hilkens Catharien M. U. , Vermeulen Hans , van Neerven R. J. Joost , Snijdewint Frank G. M. , Wierenga Eddy A. , Kapsenber Martien L.
Published: March 2007

Prostaglandin E2 (PGE2) favors T helper type 2 (Th2)‐like cytokine secretion profiles in murine and human CD4+ T cells by inhibiting the production of the Th1‐associated cytokines interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) and up‐regulating the production of the Th2‐associated cytokines IL‐4 and IL‐5 in a dose‐dependent way. However, the potent inhibition of IL‐2 production by PGE2 seems to be in contrast with the simultaneous up‐regulation of IL‐4 and IL‐5 production, because the induction of these cytokines requires IL‐2. We, therefore, investigated to which extent the net modulatory effect of PGE2 is determined by the availability of IL‐2. To this aim, we examined the effects of PGE2 on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL‐2 production. The differential modulation of Th1 and Th2 cytokines by PGE2 was observed only upon modes of stimulation resulting in high IL‐2 production. When IL‐2 production was low, PGE2 inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL‐2 availability, because (i) neutralizing antibody to IL‐2 abrogated the up‐regulatory effect of PGE2 on IL‐4 and IL‐5 secretion in experiments with high endogenous IL‐2 levels, (ii) lack of differential cytokine modulation by PGE2 in conditions with low levels of endogenous IL‐2 could be restored with exogenous IL‐2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE2 on the cytokine secretion profile of T cells critically depends on the availability of IL‐2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up‐ or down‐regulated by PGE2 under different conditions.
Cross‐reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex‐restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire
Authors: Kohler Jochen , Martin Stefan , Pflugfelder Ulrike , Ruh Helga , Vollmer Jörg , Weltzien Hans Ulrich
Published: March 2007

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)‐restricted, hapten‐specific, CD4+ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten‐conjugated, MHC class II‐associated peptides. This study for the first time directly demonstrates that hapten‐peptides account for the majority of determinants recognized by trinitrophenyl (TNP)‐specific CD4+ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP‐modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I‐Ab or from λ represser with specificity to I‐Ad as well as TNP‐proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II‐restricted hapten determinants for a number of TNP‐specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP‐specific helper T cells may cross‐react with different TNP‐peptides bound to identical class II molecules. Chemical treatment of antigen‐presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I‐Ab/TNP‐specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non‐self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP‐peptide determinants defined by us as immuno‐dominant are responsible for the induction of contact sensitivity to haptens.
Enhanced tumor necrosis factor suppression and cyclic adenosine monophosphate accumulation by combination of phosphodiesterase inhibitors and prostanoids
Authors: Sinha Bhanu , Semmler Jan , Eisenhut Tobias , Eigler Andreas , Endres Stefan
Published: March 2007

We investigated cooperative effects of phosphodiesterase (PDE) inhibitors and prostanoids on cyclic adenosine monophosphate (cAMP) accumulation and tumor necrosis factor (TNF)‐α synthesis in human peripheral blood mononuclear cells (PBMC). PDE inhibitors alone induced only a small increase in cAMP levels in lipopolysaccharide (LPS)‐stimulated PBMC. Cicaprost (a stable analogue of prostacyclin) and pentoxifylline added simultaneously to LPS‐stimulated PBMC (2.0 × 106/ml) induced a rapid increase of cAMP to a level of 100 nM that peaked within 10 min and remained at a plateau for up to 4 h. Thus combined prostanoids and PDE inhibitors enhanced cAMP accumulation. TNF‐α suppression in the presence of pentoxifylline and prostanoids exceeded that of either drug alone. The potency of different PDE inhibitors (theophylline, pentoxifylline, penthy‐droxifylline, albifylline, torbafylline, A 802715, amrinone and rolipram) to increase cAMP levels in combination with cicaprost was evaluated after 1 h of incubation. The dose‐dependent increase of cAMP for all PDE inhibitors tested in this combined stimulation provided a useful tool for evaluating the potency of PDE inhibitors on cAMP accumulation. The effective concentration of PDE inhibitors, which raised cAMP levels to 300% of control, (EC300), correlated with the IC50 for TNF‐α suppression (r = 0.930, p = 0.007, with theophylline excluded from the analysis). Interestingly, by contrast, the specific type IV PDE inhibitor rolipram caused only a moderate rise of accumulated cAMP in the same cells. Our data support cAMP as an essential mediator for TNF‐α suppression by PDE inhibitors. Furthermore, an enhanced inhibiting effect on TNF‐α production may prove therapeutically advantageous. It may occur in inflammatory and infectious diseases in vivo, since high levels of endogenous prostaglandins are liberated in these conditions.
Selective enhancing effect of the Yaa gene on immune responses against self and foreign antigens
Authors: Fossati Liliane , Iwamoto Masahiro , Merino Ramón , Izui Shozo
Published: March 2007

The BXSB Y chromosome‐linked mutant gene, Yaa, accelerates the progression of a lupus‐like autoimmune syndrome only in mice that are predisposed to autoimmune diseases. Unlike the lpr gene, which causes the defects in the Fas antigen‐mediating apoptosis, the autoimmune enhancing activity of the Yaa gene is selective, depending on autoantigens, and varies among lupus‐prone mice. To obtain a better definition of the role of the Yaa gene in the acceleration of autoimmune disease, we have investigated immune responses to several foreign antigens to determine whether the Yaa gene is able to potentiate immune responses to foreign antigens in a selective manner. We report here that the Yaa gene potentiated immune responses against foreign antigens only in mice which are genetically (H‐2‐linked) low responding, but not high or non‐responding. Moreover, studies on Yaa+‐Yaa− double bone marrow chimeric mice revealed that B cells from Yaa+ mice were selectively stimulated to produce antibodies to low‐responding antigen, human IgG, while both B cell populations similarly responded to high‐responding antigen, ovalbumin. Our results suggest that first, the selective immune enhancing activity of the Yaa gene may be related to differences in the capacity of T helper cells specific for given self or foreign antigens; and second, a specific cognate interaction of T helper cells with Yaa+ B cells is apparently responsible for the selective enhancement of immune responses to antigens, to which mice are genetically low responding.
Recombinant CD4‐IgE, a novel hybrid molecule, inducing basophils to respond to human immunodeficiency virus (HIV) and HIV‐infected target cells
Authors: Krauss Susanne , Kufer Peter , Federle Christine , Tabaszewski Pjotr , Weiss Elisabeth , Rieber E. Peter , Riethmüller Gert
Published: March 2007

Basophils and mast cells, as the main effector cells in IgE‐mediated type I hypersensitivity, are involved in the elimination of parasites and, according to recent findings, may also play an important role in the defense against bacterial and viral infections. Using a genetic engineering approach we wanted to redirect this potent IgE‐mediated defense system against intruding human immune deficiency virus. We constructed a recombinant CD4‐IgE molecule, consisting of the two N‐terminal domains of CD4 and the CH2–4 domains of the IgE heavy chain, thus providing the IgE with specificity for the gp120 of human immunodeficiency virus (HIV). The binding properties of hybrid CD4‐IgE to the high‐affinity receptor for IgE (FcεRI) on basophils as well as to the low‐affinity receptor (FcεRII or CD23) for IgE on lymphoid cells were found to be similar to those of native IgE. At the same time, the CD4 domains of the recombinant molecule retained the gp120 binding specificity with an affinity similar to that of the native CD4. By functional tests, we demonstrated that CD4‐IgE armed basophils can be triggered by free HIV and by HIV‐infected cells to release their mediators. We further show that HIV‐triggered basophils lead to a decreased replication of HIV in susceptible T cells. We, therefore, conclude that the type I hypersensitivity effector cells can be engaged in the elimination of HIV‐infected cells, at least in vitro. Because of the strong binding of the CD4‐IgE construct to the FcεRI, we assume that CD4‐IgE has a short t1/2 in serum, but may similarly to IgE exhibit prolonged resident time on basophils and mast cells, which are located close to mucosal surfaces or in the connective tissue. Thus CD4‐IgE could play an important role in the elimination of HIV also in vivo.
Mouse interleukin‐12 (IL‐12) p40 homodimer: a potent IL‐12 antagonist
Authors: Gillessen Silke , Carvajal Daisy , Ling Ping , Podlaski Frank J. , Stremlo Donna L. , Familletti Philip C. , Gubler Ueli , Presky David H. , Stern Alvin S. , Gately Maurice K.
Published: March 2007

Interleukin‐12 (IL‐12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide‐bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL‐12 (moIL‐12) p40‐transfected COS cells could inhibit IL‐12‐dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide‐linked p40 dimer [(p40)2]. The (p40)2 was 25‐ to 50‐fold more active than the p40 monomer in causing specific, dose‐dependent inhibition of IL‐12‐induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL‐12‐induced interferon‐λ (IFN‐λ) secretion by mouse splenocytes and IL‐12‐dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL‐12 in competing with 125I‐labeled moIL‐12 ([125I]moIL‐12) for binding to mouse Con A blasts. However, in contrast to moIL‐12, mouse (p40)2 displayed little ability to compete with 125I‐labeled human IL‐12 (huIL‐12) for binding to high‐affinity IL‐12 receptors (IL‐12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL‐12‐induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL‐12 in competing with [125I] huIL‐12 for binding to COS cells transfected with the human IL‐12R β subunit and expressing low‐affinity IL‐12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL‐12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL‐12 interacts with the β subunit of IL‐12R, and (iii) (p40)2 may be a suitable IL‐12 antagonist for studying the role of IL‐12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL‐12 activity.
Macrophage inflammatory protein‐1α influences eosinophil recruitment in antigen‐specific airway inflammation
Authors: Lukacs Nicholas W. , Strieter Robert M. , Shaklee Christine L. , Chensue Stephen W. , Kunkel Steven L.
Published: March 2007

Allergic airway inflammation is characterized by peribronchial eosinophil accumulation within the submucosa of the airway of the lung. In the present study we have utilized a model of airway inflammation induced by intratracheal challenge with parasite (Schistosoma mansoni) egg antigen (SEA) in presensitized mice. The recruitment of neutrophils and eosinophils into the airway was found to be maximal at 8 and 48 h post challenge, respectively. Since macrophage inflammatory protein‐1α (MIP‐1α) has previously been found to be chemotactic for eosinophils, in vitro, we postulated that MIP‐1α was involved in the airway inflammation and more specifically in eosinophil recruitment into the airway. Initial studies demonstrated an increase in MIP‐1α mRNA expression at 8 h post‐SEA challenge, as compared to vehicle‐treated control mice. We next demonstrated a significant increase in MIP‐1α protein in the lungs of SEA‐challenged mice at 8 h compared to control challenged mice, correlating to the mRNA data. Immunohistochemical staining of lungs from SEA‐challenged mice demonstrated MIP‐1α protein expression in airway epithelial cells, alveolar macrophages and in recruited mononuclear cell populations. Immunolocalization of MIP‐1α to cells within the bronchoalveolar lavage fluid demonstrated that macrophages and eosinophils stained positive for the protein. To determine the contribution of MIP‐1α expression to eosinophil accumulation, SEA‐challenged mice were passively immunized with either neutralizing MIP‐1α antibodies or normal rabbit IgG, 3–4 h prior to the intratracheal SEA challenge. These studies demonstrated a > 50% decrease in eosinophil recruitment to the lungs and airway in animals receiving neutralizing MIP‐1α antibodies with no effect on early neutrophil recruitment. These results suggest that the production of MIP‐1α, induced by an antigen‐specific response, plays an important role in recruitment of eosinophils in this airway model of inflammation.
Immunoglobulin heavy chain germ‐line JH−Cμ transcription in human precursor B lymphocytes initiates in a unique region upstream of DQ52
Authors: Thompson Allan , Timmers Erik , Schuurman Ruud K. B. , Hendriks Rudolf W.
Published: March 2007

From human precursor B cells which had both immunoglobulin (Ig) heavy (H) chain loci in germ‐line configuration, various IgH chain germ‐line transcripts were isolated and sequenced. These transcripts were shown to contain sequences derived from the JH region, the IgH chain enhancer element or the Ig switch region. A number of isolated cDNA clones contained sequences at their 5′ end that were derived from a single exon located just upstream of DQ52, designated the μ° element. Sequence analysis of a 920‐bp genomic DNA segment, containing the μ° exon and its 5′ flanking region, revealed the presence of various conserved motifs for DNA‐binding proteins, such as E2A, Ets, NF‐χB and AP‐2, which have previously been found in the IgH and L chain enhancers. We propose that the activity of μ° element, resulting in germ‐line transcription of the DQ52‐JH gene segment, is required to generate full accessibility for the V(D)J recombinase.
Lymphocytic CD43 and CD45 bear sulfate residues potentially implicated in cell to cell interactions
Authors: Giordanengo Valérie , Limouse Martine , Peyron Jean‐François , Lefebvre Jean‐Claude
Published: March 2007

CD43 is a major heavily glycosylated lymphocyte surface molecule. It has been shown to play an important role in lymphocyte activation and cell‐cell interactions. Here we demonstrate that in human activated lymphocytes and CEM T cells, CD43 is a sulfated molecule. We also observed that CD45, another lymphocyte surface glycoprotein, is a sulfated molecule. 35SO incorporation would thus appear to be an appropriate labeling method for CD43 and CD45 visualization. Moreover, we show that the level of cell surface protein sulfation can modulate CD43‐mediated homotypic aggregation induced by CD43 monoclonal antibodies. It is well known that glycoprotein sulfation is required for various recognition phenomena. Since there are numerous potential sulfation sites on CD43 and CD45, these residues could play an important role in regulating cell‐cell interactions.
The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition
Authors: Panka David J. , Salant David J. , Jacobsonn Bruce A. , Minto Andrew W. , Marshak‐Rothstein Ann
Published: March 2007

MRL/lpr mice spontaneously develop a lupus‐like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cyroprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non‐cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non‐cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site‐directed mutagenesis. The more positive residues at VH 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non‐cryoglobulins. The results show that VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.
Decay‐accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement
Authors: Marschang Peter , Sodroski Joseph , Würzner Reinhard , Dierich Manfred P.
Published: March 2007

HIV‐1 in contrast to animal retroviruses, is not lysed by human complement, but is readily inactivated by the sera from different animal species. To identify a possible species‐specific protection mechanism, HIV‐1 was expressed in cells of non‐human origin. Recombinant HIV‐1 virions that could encode the chloramphenicol acetyltransferase (CAT) protein were produced in African green monkey COS‐1 cells, mink cells and, as a control, in human HEp‐2 cells and were then used to infect CD4‐positive target cells. Analysis of the CAT activity of the target cells' revealed that fresh HIV‐1‐negative human serum reduced the infectivity of HIV‐1 derived from monkey and mink cells five‐ to tenfold, but had no effect on HIV‐1 produced in human cells. In addition, human serum efficiently lysed HIV‐1 produced in non‐human cells in contrast to HIV‐1 originating from human cells, suggesting lysis as an important mechanism of virus inactivation. Mammalian cells are protected against lysis by homologous complement by membrane‐bound regulatory molecules. Two of these complement inhibitors, namely decay‐accelerating factor (DAF) and, to a lesser extent, CD59 were found on the surface of HIV‐1 virions by means of a virus capture assay. Antibodies against DAF, but not against other host cell molecules found on the viral surface, efficiently blocked the resistance of HIV‐1 produced in human cells to human complement. These results suggest that the acquisition of DAF during the budding process from human cells protects HIV‐1 in a species‐specific way against the attack of human complement.
Expression of interleukin‐2 receptor γ on human monocytes: characterization of lineage specific post‐translational modifications
Authors: Epling‐Burnette P. K. , Wei Sheng , Liu Jin Hong , Pericle Federica , Ussery Dewayne , Russell Sarah M. , Leonard Warren J. , Djeu Julie Y.
Published: March 2007

Functional interleukin‐2 receptors (IL‐2R) on lymphocytes contain both IL‐2R β and γ chains. Whereas constitutive expression of IL‐2Rβ has been found on monocytes, the expression of IL‐2Rγ on these phagocytes has not been examined. We performed reverse‐transcription‐polymerase chain reaction with Southern blot analysis on RNA derived from purified human monocytes and discovered that they constitutively produce IL‐2Rγ mRNA. Western immunoblotting revealed 58‐and 64‐kDa forms of IL‐2Rγ on YT‐1 and human monocytes, whereas 58‐, 64‐, and 69‐kDa bands were detected using peripheral blood mononuclear cells and non‐adherent lymphocytes. These different forms resulted from variable N‐linked glycosylation since culture of the cells in tunicamycin resulted in detection of a single 39‐kDa band which corresponds to the molecular weight predicted from the deduced amino acid sequence. By co‐immunoprecipitation, the IL‐2Rβ subunit associates with only the 64‐kDa IL‐2Rγ protein band in monocytes.
Stimulation of χ transcription by a decamer‐dependent, synergistic mechanism
Authors: Sigvardsson Mikael , Bemark Mats , Leanderson Tomas
Published: March 2007

The intact SP6 χ promoter stimulated transcription 30 times more efficiently than did a control promoter consisting of a TATA motif as the only promoter element. Mutation of the SP6 χ promoter decamer in two positions reduced the transcriptional stimulation activity by over 90%. Promoters containing the SP6 χ promoter octamer or a consensus octamer in front of a TATA box were ineffective immunoglobulin promoters and stimulated at the most 15% of maximal transcription. Identical results were obtained after transfection of untransformed mouse splenic B cells stimulated by lipopolysaccharide, that express high levels of Oct2A, or of S194 cells that express negligible levels of Oct2A. Selective mutations in the penta‐decamer (pd), χY or early B cell factor (EBF) elements of the promoter reduced transcriptional stimulation by 20–30% in untransformed B cells. In S194 plasmacytoma cells the EBF mutation was functionally silent while the χY and pd mutations reduced transcriptional activation by 60‐70% in this cell line. A mutation in a TATA‐proximal E‐box motif did not alter the functional activity of the promoter in either cell population. It can be concluded that χ promoter function is highly dependent on complex interactions between individual promoter elements and that the decamer motif is pivotal for these interactions. The relative functional activity of a given promoter varied according to the target cell population used for the functional assay.
Individual differences in the orientation of the cytolytic T cell response against mouse tumor P815
Authors: Brichard Vincent G. , Warnier Guy , van Pel Aline , Morlighem Gregory , Lucas Sophie , Boon Thierry
Published: March 2007

We reported previously that the mouse tumor P815 expresses four distinct antigens (A, B, C, D) recognized by syngeneic cytolytic T lymphocytes (CTL). A fifth P815 antigen (E) was identified by means of a CTL clone derived from tumor‐infiltrating lymphocytes. We compared a number of mice for the orientation of their CTL response with respect to the various P815 antigens. Lymphocytes from mice inoculated subcutaneously with living P815 cells were stimulated in vitro with tumor cells and the resulting CTL were tested against targets expressing either antigens A and B or antigens C, D and E. Many mice had an asymmetrical response, some producing CTL directed almost exclusively against antigens A, B and others producing CTL directed almost exclusively against C, D. E. When mice were inoculated into two separate sites, different orientations in the responses of the two local lymph nodes were often observed, suggesting that individual differences in the orientation of the anti‐P815 CTL response do not result from preexisting differences between the animals. Asymmetrical CTL responses persisted in mice that were given a second injection of tumor cells. A possible interpretation of our results is that the major component of the CTL response is made of the progeny of a very small number of CTL precursors that happen to be the first to be stimulated by the tumor antigens.
Involvement of interleukin (IL)‐13, but not IL‐4, in spontaneous IgE and IgG4 production in nephrotic syndrome
Authors: Kimata Hajime , Fujimoto Masayuki , Furusho Kenshi
Published: March 2007

Nephrotic syndrome (NS) is a renal disease characterized by proteinuria and hypoalbuminemia. In NS patients without any allergic disease, serum IgE and IgG4 levels were selectively increased, and peripheral blood mononuclear cells (MNC) spontaneously produced IgE and IgG4. T cells produced interleukin (IL)‐13 spontaneously, and B cells constitutively expressed IL‐13 receptors (IL‐13R). In addition, T cells stimulated surface IgE‐negative (sIgE−) and sIgG4− B cells to produce IgE and IgG4, respectively, and IgE and IgG4 production was specifically blocked by anti‐IL‐13 antibody (Ab). MNC from atopic dermatitis (AD) patients also produced IgE and IgG4 spontaneously. However, in AD patients, T cells spontaneously produced IL‐4, but not IL‐13, and B cells constitutively expressed IL‐4R, but not IL‐13R. T cells stimulated sIgE− and sIgG4− B cells to produce IgE and IgG4, respectively, and the production was specifically blocked by anti‐IL‐4 Ab. On the other hand, sIgE+ and sIgG4+ B cells from both NS and AD patients spontaneously produced IgE and IgG4, respectively, and this production was not affected by T cells, anti‐IL‐4 Ab, or anti‐IL‐13 Ab. These results indicate that IL‐13 is involved in the enhanced production of IgE and IgG4 in NS, while IL‐4 is involved in these responses in AD.
Emergence in Cx knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant x light chain region
Authors: Rueff‐Juy Dominique , Sanchez Pierre , Faure Mathias , Drapier Anne‐Marie , Cazenave Pierre‐André
Published: March 2007

Allotype‐ or idiotype‐specific CD4+ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide‐specific CD8+ T cells. We have therefore investigated whether T‐depleted spleen cells from Ig x light chain‐expressing 129/Sv mice (129x+/+) could induce, in Cx knockout mice (129 x−/−), the generation of Ig constant x light chain region (Cx)‐specific cytotoxic T lymphocytes (CTL). The determination of TCRβ chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole Cx sequence, show that the B cells from x+/+ mice are able to elicit in Cx knockout mice, the emergence of a diverse CTL repertoire that recognizes one single Cx peptide presented by the H‐2Kb class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own x light chains.
T cells with dual antigen specificity in T cell receptor transgenic mice rejecting allografts
Authors: Simpson Elizabeth , Chandler Phillip , Sponaas Anna , Millrain Margaret , Dyson P. Julian
Published: March 2007

Allelic exclusion of T cell receptor (TCR) genes is incomplete: a significant percentage (10–30%) of normal human and mouse peripheral T cells express two surface TCR α chains, and a small percentage of peripheral human T cells have been reported to express two surface TCR β chains. A proportion of thymocytes in TCR transgenic mice rearrange endogenous T cell receptor genes, and peripheral T cells with two TCR α chains, transgenic and endogenous, have been reported. T cell clones with more than a single TCR heterodimer on their surface might be expected to show specificity for more than one cognate antigen: we report here a T cell clone with dual antigen specificity, isolated from an F5 TCR influenza nucleoprotein (NP 366–374/Db)‐specific transgenic female mouse which had rejected an H‐2‐matched male skin graft. It was selected in vitro by stimulation with male H‐2b spleen cells in the absence of the NP366–374 peptide but has specificity for both H‐Y/Db and NP366–374. This contrasted with the single NP366–374/Db specificity shown by a control clone isolated from a Rag1–/– F5 mouse. The dual antigen specificity was associated with the rearrangement of endogenous TCR genes and cell surface expression of these as well as the TCR transgene.
Altered CD40 ligand induction in tolerant T lymphocytes
Authors: Bowen Frank , Haluskey Joyce , Quill Helen
Published: March 2007

CD40 ligand (CD40L) is a member of the tumor necrosis factor superfamily and is expressed on the surface of activated T lymphocytes. The interaction of CD40L with CD40 on B cells results in B cell activation, immunoglobulin (Ig) secretion and Ig class switching. To study anergy as a mechanism of murine CD4 T cell tolerance, we determined both in vivo and in vitro that CD3‐activated anergic cells are deficient in the ability to stimulate B cell proliferation, and that anergic cells are defective for the T cell receptor/CD3‐mediated induction of CD40L expression. These results have implications for the recruitment of B cell responses by anergic T cells in vivo.
Differential binding of human interleukin‐1 (IL‐1) receptor antagonist to natural and recombinant soluble and cellular IL‐1 type I receptors
Authors: Svenson Morten , Nedergaard Susanne , Heegaard Peter M. H. , Whisenand Teri D. , Arend William P. , Bendtzen Klaus
Published: March 2007

A recently described factor, interleukin‐1 receptor antagonist binding factor (IL‐IraBF), in serum of normal individuals is immunologically related to the interleukin‐1 receptor type I (IL‐1RI). It is presumably a soluble form of the receptor that binds exclusively to interleukin‐1 receptor antagonist (IL‐1ra). Recombinant soluble human IL‐1RI expressed in COS cells (sIL‐1RI) consists of the extracellular part of the receptor and binds all three known IL‐1 species but preferentially to IL‐1ra. We further characterized the sizes and binding of IL‐1raBF and sIL‐1RI to IL‐1ra by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N‐glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL‐1ra. We also evaluated the binding of IL‐1ra to cellular IL‐1RI on MRC5 fibroblasts and the interference afforded by the soluble receptors. The results show that the protein backbones of IL‐1raBF and sIL‐1RI are of similar size (≈ 35–40 kDa) and that there are differences in the glycosylation of the two molecules. These carbohydrates were necessary for optimal binding of both molecules to IL‐1ra. Both factors blocked binding of IL‐1ra to cellular IL‐1RI, as did mAb to IL‐1ra, but the sites on IL‐1ra which bound to the mAb, and to IL‐1raBF and sIL‐1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble IL‐1RI and that IL‐1ra binds differently to these molecules and to cellular IL‐1RI.
Protective role of γ/δ T cells and α/β T cells in tuberculosis
Authors: Ladel Christoph H. , Blum Carmen , Dreher Anja , Reifenberg Kurt , Kaufmann Stefan H. E.
Published: March 2007

Tuberculosis is a chronic infectious disease which causes major health problems globally. Although acquired resistance crucially depends on α/β lymphocytes, circumstantial evidence suggests that, in addition, γ/δ T lymphocytes contribute to protection against tuberculosis. We have studied Mycobacterium tuberculosis infection in TcR‐δ‐/‐ or TcR‐β‐/‐ gene deletion mutants which completely lack γ/δ T cells or α/β T cells, respectively. Low inocula of M. tuberculosis led to death of TcR‐β‐/‐ mice and transient disease exacerbation in TcR‐δ‐/‐ mutants. Infection with higher inocula caused rapid death of TcR‐δ‐/‐ mice. The development of and bacterial containment in granulomatous lesions was markedly impaired in TcR‐β‐/‐, and less severly affected in TcR‐δ‐/‐ mutants. Mycobacteria‐induced IFN‐γ production by spleen cells in vitro was almost abolished in TcR‐β‐/‐ and virtually unaffected in TcR‐δ‐/‐ mice. Our data confirm the crucial role of α/β T cells in protection against established tuberculosis and formally prove a protective role of γ/δ T cells in early tuberculosis.
Oxpentifylline inhibits tumor necrosis factor‐α mRNA transcription and protects against arthritis in mercuric chloride‐treated Brown Norway rats
Authors: Kiely Patrick D. W. , Gillespie Kathleen M. , Oliveira David B. G.
Published: March 2007

The phosphodiesterase inhibitor oxpentifylline (OXP) has a number of potentially important immunomodulatory actions which include a selective inhibition of the Th1 subset of CD4+ cells in vitro and inhibition of tumor necrosis factor (TNF)‐α mRNA transcription. In vivo, it has a dramatic protective effect against experimental allergic encephalomyelitis. In this animal model, tissue injury is associated with both a Th1 response and with TNF‐α production, either of which could be targets for the protective action of OXP. In an attempt to clarify the relative importance of the Th cell subsets and TNF‐α in pathogenesis, we investigated the effect of OXP on a Th2 model of T cell‐dependent disease, mercuric chloride (HgCl2)‐induced autoimmunity in the Brown Norway rat. The effects of OXP on the Th1: Th2 response, TNF‐α mRNA transcription in spleen and ankle joints, and on the incidence and severity of arthritis and cecal vasculitis have been examined and the effects in vivo have been compared with those of a soluble TNF receptor‐IgG1 fusion protein (sTNFR) that neutralizes rat TNF‐α. In two separate experiments, OXP significantly enhanced unstimulated levels of splenic interleukin‐4 (IL‐4) mRNA (median 62 %, of an artificial IL‐4 mRNA construct, vs. 36.5 % in controls) and in one experiment, exaggerated the total IgE response to HgCl2. OXP inhibited HgCl2‐induced TNF‐α mRNA transcription in spleen and ankle joints. In three separate experiments, OXP had a significant protective effect against arthritis, with the mean incidence reduced from 100 % to 30 % and mean peak score reduced from 7.2 to 2.59 (experiments 1 and 2). The protection against arthritis was indistinguishable from that produced by sTNFR. There was no such protection against cecal vasculitis with either OXP or sTNFR. These results demonstrate that OXP induces a shift towards a Th2 response, inhibits TNF‐α mRNA transcription locally in joint and systemically in spleen, and has a protective effect against arthritis similar to that produced by sTNFR in the HgCl2‐treated BN rat. We conclude that TNF‐α is a critical cytokine in the pathogenesis of arthritis but not cecal vasculitis in this model, and that inhibition of TNF‐α transcription is the most important mode of action of OXP in this situation. OXP may be a potential therapeutic agent in the treatment of other arthritides, such as human rheumatoid arthritis, in which TNF‐α has been implicated in pathogenesis.
Ligation of either CD2 or CD28 rescues CD4+ T cells from HIV‐gp120‐induced apoptosis
Authors: Tuosto Loretta , Piazza Cinzia , Moretti Simona , Modesti Andrea , Greenlaw Roseanna , Lechler Robert , Lombardi Giovanna , Piccolella Enza
Published: March 2007

Temporal or quantitative imbalance in signals delivered to T cells via T cell antigen receptor (TCR), the CD4 co‐receptor, and accessory molecules can lead to anergy, apoptosis, or both. This has been observed following ligation of CD4 by HIV gp120 prior to TCR occupancy. The ability of molecules such as CD2 and CD28, interacting with their ligands LFA‐3 and B7, to provide signals that protect T cells from the induction of anergy, has been reported. Here, we demonstrate that ligation of CD2 and CD28 in conjunction with TCR occupancy rescue T cells that have been programmed for apoptotic death by prior CD4 ligation to gp120. This appears to be the result of augmented interleukin‐2 and interleukin‐4 release by the T cells following these molecular interactions. In conclusion, our results suggest that an impairment of antigen‐presenting accessory cell functions could favor gp120‐mediated apoptosis in HIV‐uninfected cells.
Listeriolysin generates a route for the presentation of exogenous antigens by major histocompatibility complex class I
Authors: Darji Ayub , Chakraborty Trinad , Wehland Jürgen , Weiss Siegfried
Published: March 2007

We have exploited the pore forming activity of listeriolysin, the hemolysin of Listeria monocytogenes, to activate CD8+ T cells with soluble proteins in vivo and in vitro. Immunization with soluble, hemolytically active listeriolysin induces both cytotoxic CD8+ T cells and CD4+ T cells, and the CD8+ T cells can be propagated with soluble listeriolysin in vitro. Moreover, conventional antigens like ovalbumin mixed together with listeriolysin are also efficiently introduced into the MHC class I pathway in vitro and in vivo. Hence, listeriolysin effectively directs itself and passenger molecules into the intracellular compartment that leads to the cytotoxic T cell response. In this way, we circumvent the bias of CD8+ T cells to recognize intracellular antigens presented by major histocompatibility complex class I molecules. As cytotoxic CD8+ T cells are of pivotal importance in eliminating viral and microbial pathogens, the findings reported here could prove to be useful in vaccine development.