bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-linking CD40 on human B cell precursors inhibits or enhances growth depending on the stage of development and the IL costimulus.
Authors: Larson A W , LeBien T W
Published: January 2023

The function of the cell surface molecule CD40 on B cell precursors (BCP) is not well understood. We now report studies using the L cell/CD40 system (anti-CD40 mAb immobilized on CD32+ mouse L cells) to assess the potential function of CD40 during human B cell ontogeny. Stimulation of human B lineage cells with IL-4 in the L cell/CD40 system yielded a hierarchy of responsiveness: high density tonsillar B cells > fetal splenic B cells > fetal bone marrow surface Ig+ immature B cells > fetal bone marrow surface Ig- BCP. Using a microsphere/flow cytometry growth quantitation assay, we found that substituting IL-3 for IL-4 in the L cell/CD40 system provided a stronger growth stimulus for fetal bone marrow BCP and immature B cells. We also found that FACS-purified fetal bone marrow CD10+/CD34+/CD40+/cytoplasmic mu- pro-B cells responded maximally to IL-3 plus IL-7. Surprisingly, anti-CD40 inhibited the pro-B cell response to IL-7. In contrast, FACS-purified fetal bone marrow CD10+/CD34-/CD40+/cytoplasmic mu+ pre-B cells were essentially nonresponsive to IL-3, IL-7, or anti-CD40 alone, but were uniquely responsive to IL-3 plus anti-CD40. B-lineage cells derived after 14 days from IL-7-stimulated pro-B cells were predominantly CD19+/L chain-, whereas pre-B cells stimulated with IL-3 plus anti-CD40 were predominantly CD19+/L chain+. The L chain+ cells from pre-B cell cultures were both mu+/delta+ and mu-/delta+. Our results demonstrate that the response to CD40 signaling depends upon the BCP developmental stage and the IL costimulus, and indicate that normal human pro-B cells and pre-B cells have different growth factor requirements.
Type XI collagen-induced arthritis in the Lewis rat. Characterization of cellular and humoral immune responses to native types XI, V, and II collagen and constituent alpha-chains.
Authors: Cremer M A , Ye X J , Terato K , Owens S W , Seyer J M , Kang A H
Published: January 2023

Autoimmune arthritis can be induced in rats by immunization with cartilage-specific types II and XI collagen. Type XI collagen is composed of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains, in which alpha 3(XI) is essentially identical to alpha 1(II) of type II, and alpha 1(XI) and alpha 2(XI) are similar to alpha 1(V) and alpha 2(V) of type V collagen. To characterize the immune response to type XI collagen, Lewis rats were injected with bovine type XI collagen, and the cellular and humoral responses were compared with those of rats injected with types V and II collagen. Arthritis, IgG deposits in cartilage, and joint destruction were seen in rats immunized with types XI and II collagen. Type XI elicited strong cellular responses to rat types XI, V, and II; conversely, types II and V collagen elicited strong responses to rat type XI. Antitype XI Abs reacted with rat type XI, moderately with rat type V, but poorly with rat type II. Direct and inhibition ELISA showed that cross-reactions between types XI and V collagen resulted from recognition of determinants shared by their respective alpha 2(XI) and alpha 1(V) chains. Abs eluted from joints of rats immunized with type XI collagen, however, reacted only with native rat type XI collagen. These data demonstrate that type XI collagen induces diverse populations of Abs differing in collagen-type specificity, and suggest that only those Abs to native rat type XI collagen are central to the pathogenesis of type XI collagen-induced arthritis.
Mutations in Ig V(D)J genes are distributed asymmetrically and independently of the position of V(D)J.
Authors: Weber J S , Berry J , Manser T , Claflin J L
Published: January 2023

To study the distribution of somatic mutations in the DNA flanking the 5' side of rearranged Ig V genes, the 5' region of 14 to 21 mutant forms of three different V(D)J were sequenced. These were compared with a fourth V(D)J for which the flanking sequences of 10 mutant forms are known. The leader intron of these four V genes varied from 82 to 365 nucleotides in length. Analysis of the data showed that: 1) The mutation frequency is distributed asymmetrically with respect to the V(D)J exon with skewing in the 3' direction. 2) The distribution of mutations 5' of V(D)J seems to be related to the size of the leader intron. The significance of these findings for models of hypermutation is discussed.
Leukocyte interactions with vascular endothelium. New insights into selectin-mediated attachment and rolling.
Authors: Ley K , Tedder T F
Published: January 2023

Among the earliest signs of inflammation is the capture of leukocytes from the blood stream and their subsequent rolling along the endothelium of postcapillary venules. This commentary summarizes recent insight into the molecular basis of leukocyte rolling gained from gene-targeted mice, Ab blocking studies, and in vitro and in vivo reconstitution assays. These data reveal how the selectins individually and collectively contribute to the process of leukocyte capture and subsequent rolling on vascular endothelium.
Differential expression of the blk and ret tyrosine kinases during B lineage development is dependent on Ig rearrangement.
Authors: Wasserman R , Li Y S , Hardy R R
Published: January 2023

Expression of Ig transgenes in recombination-deficient mutant scid and Rag-1-mice results in the generation of pre-B and B cells, which are normally absent from these animals. In screening for protein tyrosine kinases (PTKs) that may play a role in this progression beyond the pro-B stage, we have identified five differentially regulated PTKs and compared their gene expression in defined stages of early B-lineage cells from normal, mutant, and Ig-transgenic mutant mice. Three PTKs (fgr, flk2/flt3, and tsk) show a comparable decrease at an early stage in all mice. In contrast, the decreasing expression of ret and the increasing expression of blk seen in differentiating B cells from normal mice are not observed in the mutant mice, unless they carry Ig transgenes. Therefore, our results show that the expression of certain PTKs is dependent on productive Ig rearrangement and suggest important roles for both Ret and Blk at distinct stages in the Ig-dependent progression of B cell differentiation.
Lipopolysaccharide-induced expression of TNF-alpha gene in the macrophage cell line ANA-1 is regulated at the level of transcription processivity.
Authors: Biragyn A , Nedospasov S A
Published: January 2023

Increasing evidence suggests that regulation of transcription at the level of elongation or processivity may be an important mechanism governing expression of eukaryotic genes. In this study we compared LPS- and IFN-gamma-induced transcription of the TNF-alpha gene in two murine macrophage cell lines, ANA1 and Pu5-1.8. Our data from nuclear run-on analysis indicate that in ANA-1 cells TNF-alpha expression is regulated at the transcriptional level, as previously found in primary macrophages. In contrast, in Pu5-1.8 cells the TNF gene is constitutively transcribed. Using several short probes spanning the TNF gene we find that in ANA-1 cells transcription can be initiated before activation, but such transcripts have low processivity and are prematurely terminated or arrested within the gene. Induction with LPS alone or with LPS plus IFN-gamma results both in increased transcription initiation, and in the increased processivity of these transcripts. In Pu 5-1.8 cells neither type of transcriptional regulation of the TNF gene is observed. Our results indicate that the TNF gene is preactivated in ANA-1 cells, and RNA polymerase is allowed to initiate transcription, but due to the low processivity of the transcripts very little mRNA is formed. After LPS stimulation the TNF gene is maximally activated both by increased initiation and by higher processivity of the transcript, and each of these components of activation do not require a new protein synthesis. Our findings are consistent with a recently proposed model that the same transcriptional activators contribute to both initiation and processivity of transcription. In the case of LPS and LPS+IFN-gamma stimulation of macrophages, inducible members of NF-kappa B/Rel family are likely candidate transcriptional activators.
Role of protein kinase C isozymes in Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.
Authors: Zheng L , Zomerdijk T P , Aarnoudse C , van Furth R , Nibbering P H
Published: January 2023

Intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma R is known to be a phospholipase C (PLC)-dependent process. Activation of PLC leads to the formation of second messengers that synergistically activate protein kinase C (PKC). The aim of this study was to obtain more insight into the role of PKC in Fc gamma R-mediated killing process. PKC inhibitors H-7 and staurosporine markedly suppressed the killing of S. aureus by monocytes stimulated by cross-linking Fc gamma RI or -II. Cross-linking Fc gamma R caused a transient increase in PKC activity in the membranes of monocytes, as measured by Ca2+/phospholipid-dependent phosphorylation of histone. Western blot analysis revealed that cross-linking Fc gamma R stimulated a transient increase in PKC-beta in the membranes of monocytes with kinetics that correlated closely with the translocation of PKC activity. Cross-linking Fc gamma R on monocytes also stimulated the translocation of PKC-epsilon but not PKC-alpha. PMA and 1-oleoyl-2-acetylglycerol (OAG), which caused translocation of PKC-alpha, -beta, and -epsilon, did not stimulate the killing process. Incubation with these PKC activators for 10 min rendered monocytes unresponsive to stimulation of killing of S. aureus via Fc gamma R. It could be that activation of certain PKC isozymes, probably PKC-alpha and -epsilon, by these activators causes feedback inhibition of PLC and, consequently, the killing in monocytes, because PMA blocks the Fc gamma R-mediated intracellular inositol(1,4,5)P3 formation and PKC translocation. Together, our results indicate that PKC isozymes play an important role in both stimulation and inhibition of the Fc gamma R-mediated intracellular killing of bacteria by monocytes.
Genetic variation among 129 substrains: practical consequences.
Authors: Sechler J M , Yip J C , Rosenberg A S
Published: January 2023

We designed a series of experiments to define the role of IFN-gamma in cellular interactions mediating graft rejection by assessing the rejection of H-Y disparate grafts in both ligand and receptor knockout mice and their control inbred strain. In the course of these studies it became apparent that neither knockout strain is histocompatible with the putative control and that the putative control is not histocompatible with either knockout strain. In the process of deducing why this might be so, it became apparent that the putative control is not an inbred strain of mouse. Thus, in the absence of rigorous genetic control, the utility of such knockout strains of mice for assessing the effects of cytokines and receptors in transplantation and autoimmunity is limited.
In vivo effects of T helper cell type 2 cytokines on macrophage antigen-presenting cell induction of T helper subsets.
Authors: Cua D J , Stohlman S A
Published: January 2023

SJL mice provide an interesting paradigm to examine the role(s) of APC in the differential induction of Th1 and Th2 cells. Immunization of young male SJL mice results in the preferential induction of Th2 cells, whereas Th1 cells are induced in age-matched female or older male SJL mice. The absence of Th1 responses in young male mice is associated with in vivo IL-4 and IL-10 down-regulating Mac-3+ APC priming of Th1 cells. The present report examines the mechanism of this APC-dependent induction of Th subsets. Examination of the surface expression of MHC class II, adhesion molecules (CD11a, CD11b, CD48, CD54, and CD102) or costimulatory molecules (CD24, CD80, and CD86) showed no differences between male- and female-derived Mac-3+ APC populations. In addition, no differences were detected in IL-1alpha, IL-1beta, IL-18, TNF-alpha, or IL-12 p35 mRNA expression. However, reduced expression of both IL-10 and IL-12 p40 mRNA were found in Mac-3+ cells from male mice compared with those in Mac-3+ cells from female mice. Anti-IL-4 or anti-IL-10 mAb treatment of young male donor mice eliminated the reduction of both IL-10 and IL-12 p40 mRNA, suggesting that the Th2 inducer phenotype is related to a decreased IL-12 secretion. Consistent with this idea, fewer IL-12 p40-secreting Mac-3+ cells were found in male mice compared with female mice, and treatment with rIL-12 resulted in the priming of Th1 cells in male mice. These data suggest that increased Th2 cytokines in vivo before encounter with Ag inhibit APC expression of IL-12, resulting in the preferential induction of Th2 cells in male SJL mice.
Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.
Authors: Levine B L , Bernstein W B , Connors M , Craighead N , Lindsten T , Thompson C B , June C H
Published: January 2023

In this report, conditions for prolonged in vitro proliferation of polyclonal adult CD4+ T cells via stimulation with immobilized anti-CD3 plus anti-CD28 have been established. CD4+ cells maintained exponential growth for more than 60 days during which a total 10(9)- to 10(11)-fold expansion occurred. Cell cultures exhibited cyclical changes in cell volume, indicating that, in terms of proliferative rate, cells do not have to rest before restimulation. Indeed, electronic cell size analysis was the most reliable method to determine when to restimulate with additional immobilized mAb. The initial approximately 10(5)-fold expansion was autocrine, occurring in the absence of exogenous cytokines or feeder cells. Addition of recombinant human IL-2 after the initial autocrine expansion resulted in continued exponential proliferation. Phorbol ester plus ionomycin also induced long-term growth when combined with anti-CD28 stimulation. Analysis of the T cell repertoire after prolonged expansion revealed a diverse repertoire as assessed by anti-TCR Vbeta Abs or a PCR-based assay. Cytokines produced were consistent with maintenance of both Th1 and Th2 phenotypes; however, the mode of CD3 and CD28 stimulation could influence the cytokine secretion pattern. When anti-CD3 and anti-CD28 were immobilized on the same surface, ELISAs on culture supernatants revealed a pattern consistent with Th1 secretion. Northern analysis revealed that cytokine gene expression remained inducible. Spontaneous growth or cell transformation was not observed in more than 100 experiments. Together, these observations may have implications for gene therapy and adoptive immunotherapy. Furthermore, these culture conditions establish a model to study the finite lifespan of mature T lymphocytes.
IgA plasma cells in vascular tissue of patients with Kawasaki syndrome.
Authors: Rowley A H , Eckerley C A , Jäck H M , Shulman S T , Baker S C
Published: January 2023

The etiology and pathogenesis of Kawasaki syndrome (KS) remain unknown. Clinical and epidemiologic features of KS are consistent with an infectious cause. To search for an etiologic agent of KS, a phage cDNA expression library was constructed from the aorto-iliac junction of a patient with fatal acute KS and screened with convalescent KS serum followed by anti-human Ig. Unexpectedly, 0.1% of the clones in the library react with anti-human Ig, indicating the presence of many Ig-producing B lymphocytes in the vasculitic tissue. To confirm this finding and to determine the isotypes produced, frozen vascular tissue sections from the patient and paraffin sections from coronary arteries from six additional patients with fatal acute or subacute KS were incubated with Abs to Ig isotypes. Histopathology of the tissues revealed the presence of many plasma cells in the inflammatory infiltrate. IgA was the predominant isotype produced in vascular tissue in all seven KS patients. IgM- and IgG-producing cells were less often detected. We conclude that there is a marked plasma cell response within the vasculitic tissue in KS, with unusual IgA production locally in this nonlymphoid, nonmucosal tissue. We suggest that the prominence of IgA plasma cells in the vascular infiltrate in the early, acute, and subacute stages of KS indicates an Ag-driven immune response to an etiologic agent with a respiratory or gastrointestinal portal of entry and speculate that this unusual immune response is integral to the pathogenesis of the illness.
IL-1 beta-converting enzyme (ICE) is present and functional in human alveolar macrophages: macrophage IL-1 beta release limitation is ICE independent.
Authors: Wewers M D , Dare H A , Winnard A V , Parker J M , Miller D K
Published: January 2023

Tissue macrophages readily produce intracellular pro-IL-1beta in response to stimuli such as LPS, but are limited in mature IL-1beta release compared with blood monocytes. The mechanism of this IL-1beta control may provide important insights into the physiology of IL-1beta at the tissue level. Since it has been hypothesized that IL-1beta processing by the IL-1beta-converting enzyme (ICE) regulates IL-1beta release, we compared human alveolar macrophages and human blood monocytes for relative ICE expression and activation. Using immunoblots and enzyme-linked immunoassay for ICE, we demonstrate that alveolar macrophages do not differ from blood monocytes in antigenic p45 ICE. Furthermore, an indirect assay for functional ICE documents similar ICE activities in both monocytes and alveolar macrophages, i.e., similar concentrations of soluble synthetic ICE inhibitor (IC50 values of 0.3 +/- 0.01 and 0.6 +/- 0.2 microM, respectively) are required to block mature IL-1beta generation. However, as has been reported for THP-1 myelomonocytic cells, neither alveolar macrophages nor blood monocytes contain directly quantifiable levels of functional ICE forms (p22/p20 and p10) when assayed by immunoblots or by a sensitive capture ELISA that uses an irreversible, biotinylated ICE inhibitor. These findings document that the macrophage limitation in mature IL-1beta release is not due to a lack of ICE or to an inability to activate ICE. Finally, using a staged release assay, the time to half-maximum mature IL-1beta release is significantly depressed in macrophages compared with that in monocytes. Taken together, these findings suggest that macrophage IL-1beta export is regulated independently of ICE activation.
Identification of shared tumor-associated antigen peptides between two spontaneous lung carcinomas.
Authors: Mandelboim O , Bar-Haim E , Vadai E , Fridkin M , Eisenbach L
Published: January 2023

CTLs recognize antigenic peptides bound to MHC class I Ags on the cell surface of tumor cells. Tumor-associated Ag (TAA) peptides are 8 to 10 amino acids long and can be derived from normal, mutated, or viral proteins. The majority of T cell-defined Ags have been identified in human melanoma cells. These were shown to be commonly expressed by different allogeneic melanomas that share the same MHC molecule. We have recently isolated Kb-restricted TAA peptides, which are mutations of the gap junction protein connexin 37, from the spontaneous C57BL/6 Lewis lung carcinoma (3LL). These peptides, named MUT 1 and MUT 2, serve as CTL epitopes and can induce CTL activity in vivo. Using CTL cross-reaction assays, peptide extraction, HPLC fractionation, and reverse transcriptase-PCR amplification, we show that clones of another spontaneous C57BL/6 lung carcinoma, CMT 64, share TAA peptides with the 3LL carcinoma. Vaccination with synthetic MUT 1 or MUT 2 induces CTLs that efficiently lyse CMT 64-derived clones, protects mice from CMT 64 metastasis, and affords therapy of established CMT 64 metastases. Hence, shared CTL epitopes exist between two spontaneous murine lung carcinomas.
Regulation of IL-18 (IFN-gamma-inducing factor) gene expression.
Authors: Tone M , Thompson S A , Tone Y , Fairchild P J , Waldmann H
Published: January 2023

IL-18 (also known as IFN-gamma-inducing factor), although structurally unrelated to IL-12, shares with it the role of activating NK cells and polarizing T cells toward Th1 cell function. To understand how the IL-18 gene (and consequently Th1 function) is regulated, we have determined the gene structure and investigated the mechanisms of transcriptional control and cell type expression. The mouse IL-18 gene comprises seven exons distributed over 26 kb. Exons 1 and 2 of this gene are 5'-noncoding exons. Promoter activity was detected upstream of these noncoding exons in two distinct regions. Both promoters are TATA-less and not G+C rich. The promoter activity located upstream of exon 2 was shown to act constitutively, while the activity located upstream of exon 1 was up-regulated in activated macrophage and T cell lines. IL-18 gene expression may be regulated in a wide range of cell types by the activities of these two distinct promoters. IL-18 is known to be synthesized as a precursor, pro-IL-18, and its maturation is controlled by IL-1beta-converting enzyme (ICE). We observed concordant expression of IL-18 and ICE mRNAs in a wide range of cell types, unlike the more restricted expression of IL-12 p40 mRNA. The widespread IL-18 mRNA distribution and the special relationship with ICE lead us to the hypothesis that IL-18 expression may be coupled with apoptotic processes involving activation of ICE or ICE-like proteinase.
Calcium-dependent neutrophil secretion: characterization and regulation by annexins.
Authors: Rosales J L , Ernst J D
Published: January 2023

To gain direct access to the secretory machinery and study the regulation, mechanisms, and effectors of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies that permeabilized neutrophils secrete in response to calcium alone, but we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 microM Ca2+ and reaches a plateau between 1.0 and 60 to 80 microM. When stimulated with >80 microM Ca2+, secretion is two- to threefold greater than at lower [Ca2+], suggesting that two distinct mechanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells is 80% that of f-met-leu-phe-stimulated intact cells, indicating that the essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization causes leakage of 100% of annexins V and VI, but 41% of annexin I and 12% of annexin III are retained. Immunofluorescence microscopy revealed that retained annexins I and III are associated with granule membranes. Addition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mechanism of secretion.
Inhibition of inducible nitric oxide synthase intensifies injury and functional deterioration in autoimmune interstitial nephritis.
Authors: Gabbai F B , Boggiano C , Peter T , Khang S , Archer C , Gold D P , Kelly C J
Published: January 2023

T lymphocytes are exquisitely sensitive to the antiproliferative effects of nitric oxide. We examined the effects of oral administration of two nitric oxide synthase inhibitors, Nw-nitro-L-arginine methyl ester (L-NAME) and L-N6-(1-iminoethyl)lysine (L-NIL), on the course of T cell-dependent autoimmune interstitial nephritis in Brown Norway rats. Kidneys from rats immunized to produce interstitial nephritis display a net generation of nitric oxide end products. By immunohistochemical staining, the cytokine-inducible nitric oxide synthase (iNOS) is expressed in cortical tubular epithelial cells. Treatment with either inhibitor results in markedly more severe disease following immunization. Animals receiving L-NAME were hypertensive, while those treated with L-NIL, a highly selective inhibitor of iNOS, were not. Evaluation of the expression of IFN-gamma, IL-2, and IL-4 in diseased kidneys by quantitative reverse transcriptase-PCR demonstrated that L-NAME-treated animals displayed significantly augmented levels of IFN-gamma and IL-2 with preserved ratios of IFN-gamma/IL-4 and IL-2/IL-4, while L-NIL-treated animals had augmented levels of IL-2 and IFN-gamma with augmented IFN-gamma/IL-4 and IL-2/IL-4 ratios. Animals treated with L-NAME or L-NIL both had augmented Ag-specific IgG responses. The L-NAME group demonstrated increases in both the IgG2a and IgG1 subtypes, with a constant IgG2a/IgG1 ratio, while the L-NIL group demonstrated an increase in the ratio of the IgG2a/IgG1 response. These Ab and cytokine data suggest that the L-NIL-treated animals had a skewing of their immune response toward a Th1-like response. We conclude that in autoimmune interstitial nephritis, generation of nitric oxide through the iNOS pathway has host-protective effects, and suggest that this may be broadly applicable to T cell-mediated pathologies.
Three domains of SLP-76 are required for its optimal function in a T cell line.
Authors: Musci M A , Motto D G , Ross S E , Fang N , Koretzky G A
Published: January 2023

We and others have shown that overexpression of SLP-76 augments TCR-stimulated IL-2 promoter activity in the Jurkat T cell line. In this report we investigate the signaling mechanisms through which SLP-76 mediates its effect on T cell activation. We show that overexpressed SLP-76 acts downstream of TCR-stimulated protein tyrosine kinases, but does not affect calcium signaling. Overexpression of SLP-76 does, however, augment TCR stimulation of both ERK (extracellular signal-regulated kinase) activity and a reporter construct driven by activating protein-1 binding sites. Structure/function analysis reveals that three distinct regions of SLP-76, each important for protein associations, are required for augmentation of TCR-induced nuclear factor-AT activity. These data suggest that SLP-76 functions as an adapter molecule that requires three unique domains to link proximal TCR signals in T cells.
Characterization of IL-12 receptor beta1 chain (IL-12Rbeta1)-deficient mice: IL-12Rbeta1 is an essential component of the functional mouse IL-12 receptor.
Authors: Wu C , Ferrante J , Gately M K , Magram J
Published: January 2023

Two chains of the IL-12R, IL-12Rbeta1 and IL-12Rbeta2, have recently been cloned. To determine the role of IL-12Rbeta1 in mediating the biologic functions of IL-12 in mice, we have generated IL-12Rbeta1-deficient (IL-12Rbeta1(-/-)) mice by targeted mutation in ES cells. Con A-activated splenocytes from IL-12Rbeta1(+/+) mice displayed both high and low affinity IL-12-binding sites, whereas Con A-activated splenocytes from IL-12Rbeta1(-/-) mice expressed only low affinity IL-12-binding sites. Consistent with the expression of low affinity IL-12-binding sites on IL-12Rbeta1(-/-) lymphoblasts, these cells expressed normal amounts of IL-12Rbeta2 mRNA. Unlike those from IL-12Rbeta1(+/+) mice, Con A-activated splenocytes from IL-12Rbeta1(-/-) mice failed to proliferate or produce IFN-gamma in response to IL-12, even at very high concentrations (67 nM). In contrast, lymphoblasts from both types of mice proliferated equally well to IL-2 or IL-7. Splenocytes from IL-12Rbeta1(-/-) mice also failed to display enhanced NK lytic activity when cultured with IL-12 but responded normally to IL-2. Similar to IL-12 p40-deficient mice, IL-12Rbeta1(-/-) mice were impaired in their ability to produce IFN-gamma in response to endotoxin administration in vivo, and IL-12Rbeta1(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or soluble anti-CD3 mAb in vitro. These results demonstrate that IL-12Rbeta1 is required for mouse T and NK cells to respond to IL-12 and that expression of low affinity IL-12-binding sites, presumably reflecting expression of IL-12Rbeta2, is by itself insufficient to mediate IL-12 responsiveness, even in the presence of very high concentrations of IL-12.
Peptide antigen or superantigen-induced down-regulation of TCRs involves both stimulated and unstimulated receptors.
Authors: Niedergang F , Dautry-Varsat A , Alcover A
Published: January 2023

T cell activation by peptide/MHC complexes, superantigens, or mAbs induces the down-regulation of cell surface TCRs. We addressed the question of whether TCR down-modulation affects only TCRs that had directly interacted with their ligand or whether down-modulation could also affect TCRs that had not interacted with their ligand. To this end, we generated T cells coexpressing equal levels of two different TCRs by transfecting the appropriate cDNAs into cells of the human T cell line, Jurkat. Each set of TCRs can be distinguished by means of anti-Vbeta mAbs and can be stimulated separately with peptide Ag, bacterial superantigens, or mAbs. We found that activation of these cells with each of these stimuli down-modulated not only directly stimulated TCR complexes but also unstimulated ones. Comodulation of stimulated and unstimulated receptors may reflect functional interactions between surface TCRs that could take place during Ag or superantigen recognition by T cells without the need for ligand cross-linking. Consistent with this idea, both stimulated and unstimulated receptors colocalized in patches on the cell surface after activation.
T cell genetic background determines maintenance of IL-12 signaling: effects on BALB/c and B10.D2 T helper cell type 1 phenotype development.
Authors: Güler M L , Jacobson N G , Gubler U , Murphy K M
Published: January 2023

In this report, we examined the molecular basis underlying the genetic difference between BALB/c and B10.D2 T cells for T helper phenotype development in vitro. We found a strain-dependent difference in early maintenance of IL-12 responsiveness by T cells developing in vitro in unmanipulated (neutral) conditions. Thus, when activated without addition of exogenous cytokines or neutralization of endogenous cytokines, B10.D2, but not BALB/c, T cells remain responsive to IL-12 when activated for 7 days. The pattern of IL-12 responsiveness correlated with expression of the IL-12R signaling subunit, IL-12R beta2, and with IL-12-induced STAT4 phosphorylation. When activated under neutral conditions, BALB/c T cells rapidly lose IL-12R beta2 expression, STAT4 phosphorylation, and functional IL-12 responsiveness. More efficient maintenance of IL-12R beta2 expression by B10.D2 T cells activated under neutral conditions may explain the previously observed increase in IFN-gamma production relative to that of BALB/c. This difference could potentially provide greater protection from certain pathogens that do not immediately elicit strong Th1-inducing conditions via activation of the innate immune system.
Differential interaction of nuclear factors with the leukocyte-specific pp52 promoter in B and T cells.
Authors: Omori S A , Smale S , O'Shea-Greenfield A , Wall R
Published: January 2023

The leukocyte-specific, cytoskeleton-binding pp52 (LSP-1, WP-34) protein is widely expressed in multiple leukocyte lineages, including B and T lymphocytes, granulocytes, and macrophages. We previously detected a tissue-specific promoter preceding the exon encoding the N terminus of the pp52 leukocyte protein. Here we describe the functional characterization of this promoter and identification of the factors in B and T cells that regulate its activity. The pp52 promoter contains an initiator specifying the unique 5' terminus of pp52 mRNA, tandem pairs of Ets and SP1 motifs, and a lone C/EBP motif. All these motifs are essential and collectively control transcriptional activity. DNA binding studies and Ab supershift assays revealed that different combinations of factors interact with these motifs in B cells vs T cells. The Ets motifs are preferentially bound by PU-1 in B cell extracts from all stages of development, whereas a different Ets family member reacts with these motifs in T cell extracts. The C/EBP motif is bound by Ig/EBP-1 in pre-B cell and T cell extracts, but is replaced by nuclear factor-IL-6beta or a nuclear factor-IL-6beta-Ig/EBP-1 heterodimer in plasmacytoma cell extracts. Despite its reported role as a negative regulator of transcription, Ig/EBP-1 appears to exert a stimulatory effect on this promoter. These findings reveal the features controlling the pp52 promoter in B and T cells and provide the foundation for determining the regulation of this promoter in other leukocyte lineages.
Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone.
Authors: Hartmeyer M , Scholzen T , Becher E , Bhardwaj R S , Schwarz T , Luger T A
Published: January 2023

Pro-opiomelanocortin (POMC)-derived peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) recently have been recognized as mediators with potent immunomodulating and anti-inflammatory properties. Their effects are mediated via different protein G-coupled melanocortin (MC) receptors that are capable to bind one or more POMC-derived peptides. Among these receptors, MC-1 is specific for alpha-MSH and adrenocorticotropin. The purpose of the present study was to investigate whether MC receptors are expressed on normal human dermal microvascular endothelial cells (HDMEC) as well as transformed human dermal microvascular endothelial cells (HMEC-1). Using semiquantitative reverse transcriptase-PCR and MC receptor-specific primers, both HDMEC and HMEC-1 were found to express MC-1 constitutively. In addition, MC-1 expression was increased upon stimulation with IL-1beta or alpha-MSH itself. Other known MC receptors were neither detectable in unstimulated nor in IL-1beta- or alpha-MSH-stimulated cells. The binding of alpha-MSH by HMEC-1 was specific and saturable as demonstrated by competitive and saturation-binding studies with 125I-labeled alpha-MSH (Kd: 1.1 nM). To evaluate the physiologic relevance of MC-1 expression, HMEC-1 were treated with various concentrations of alpha-MSH (10(-15)-10(-6) M) and were investigated for their cytokine-producing capacity. Alpha-MSH (10(-10)-10(-8) M) significantly up-regulated IL-8 release and mRNA expression by HMEC-1. In contrast, the production of IL-1 or IL-6 by HMEC-1 was not affected upon treatment with alpha-MSH. These data provide first evidence that HDMEC express functional MC receptors. Therefore, alpha-MSH, which is released in the skin during cutaneous inflammation via inducing chemokines may represent an important signal required for leukocyte-endothelial cell interaction.
Selective conversion of big endothelins to tracheal smooth muscle-constricting 31-amino acid-length endothelins by chymase from human mast cells.
Authors: Nakano A , Kishi F , Minami K , Wakabayashi H , Nakaya Y , Kido H
Published: January 2023

Chymase from human mast cells selectively cleaved big endothelins (ETs) at the Tyr31-Gly32 bond and produced novel trachea-constricting 31-amino acid-length endothelins, ETs(1-31), without any further degradation products. Chymases from other species, such as the enzymes from rat connective tissue and mucosal mast cells, and the other chymotrypsin-like proteases examined degraded big ETs. ETs(1-31) exhibited various contractile potencies as to the rat trachea in comparison with 21-amino acid-length endothelins, ETs(1-21), and big ETs: ET-1(1-21) > ET-1(1-31) > big ET-1; ET-2(1-31) > ET-2(1-21) > or = big ET-2; ET-3(1-21) > or = ET-3(1-31) > or = big ET-3. Among the ETs(1-31), ET-2(1-31) was the most potent constrictor, its potency being similar to that of ET-1(1-21) and stronger than that of ET-2(1-21). The contractile activity of ETs(1-31) may not be the consequence of conversion to the corresponding ETs(1-21) by phosphoramidon-sensitive ET-converting enzymes or other chymotrypsin-type proteases and metalloendopeptidases, because the contractile activity was not inhibited significantly on treatment with inhibitors of these proteases before the addition of ET-1(1-31). Inhibitors of chymotrypsin-type serine proteases, on the contrary, significantly enhanced the contractile activity exhibited by ET-1(1-31) and big ET-1, but not that by ET-1(1-21). These results suggest that protease(s) on the surface of the rat trachea tends to degrade ETs(1-31) and big ETs, and thereby reduces their contractile activity. Taken together, the results suggest that trachea-constricting ETs(1-31) generated by human chymase may play a role in the hyper-responsive airway in allergic inflammation.
Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins.
Authors: Ishii K , Amagai M , Hall R P , Hashimoto T , Takayanagi A , Gamou S , Shimizu N , Nishikawa T
Published: January 2023

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1, respectively. Routine immunofluorescence testing of skin and serum from patients cannot distinguish between these two severe diseases since both have IgG Abs directed against keratinocyte cell surfaces. In this study, recombinant Dsg3 and Dsg1, produced as secreted proteins by baculovirus expression, have been utilized to develop ELISAs for the specific characterization of their autoantibodies. Of 49 PV sera, 46 were positive in the Dsg3 ELISA and 44 of 46 PF sera were positive in the Dsg1 ELISA, compared with only 3 of 23 sera of bullous pemphigoid, and none of 53 normal control sera in both ELISAs. Both the Dsg3 and Dsg1 ELISAs were more specific and sensitive than conventional immunofluorescence staining. These Ag-specific ELISAs revealed that more than one-half of PV sera (26 of 49) had anti-Dsg1 Abs in addition to anti-Dsg3 Abs. PV patients who had not only oral mucous lesions but also significant skin involvement tended to have higher titers of anti-Dsg1 Abs. Furthermore, the ELISA reactivity correlated well with clinical disease activity in 5 of 6 PV and 5 of 5 PF patients. This ELISA provides a sensitive and highly specific assay for the diagnosis of patients with PV and PF, the correlation of disease activity with serum Ab levels, and a novel tool for investigating the immunopathogenesis of pemphigus.
HIV-1 envelope-specific CD4+ T helper cells from simian/human immunodeficiency virus-infected rhesus monkeys recognize epitopes restricted by MHC class II DRB1*0406 and DRB*W201 molecules.
Authors: Lekutis C , Letvin N L
Published: January 2023

The HIV-1 envelope (Env)-specific Th cell response of rhesus monkeys infected with nonpathogenic chimeric simian/human immunodeficiency viruses (SHIV) was assessed at a molecular level by establishing rgp120-specific CD4+ T cell lines. Epitopes recognized by these MHC class II-restricted Th cells were identified within the second (C2), third (C3), and fifth (C5) conserved regions of HIV-1 gp120. The epitope located in C2 was found to be restricted by the rhesus monkey (Macaca mulatta) leukocyte Ag Mamu-DRB1*0406, and the Th cell epitope in C5 was found to be restricted by Mamu-DRB*W201. The restriction element requirements of the C2 and C5 epitope-specific CD4+ Th cells appear to be rather stringent, because these peptide epitopes were not recognized in the presence of other Mamu-DR expressing cell lines. The ability to analyze HIV-1 Env-specific CD4+ T cell responses in SHIV-infected monkeys will enhance the utility of this model for studying AIDS pathogenesis and for assessing novel HIV-1 vaccine strategies.
Affinity Maturation in Lyn Kinase-Deficient Mice with Defective Germinal Center Formation
Authors: Kato Jun , Motoyama Noboru , Taniuchi Ichiro , Takeshita Hiromichi , Toyoda Masaki , Masuda Keiji , Watanabe Takeshi
Published: January 2023

Lyn kinase-deficient (lyn−/−) mice show several abnormalities such as reduced numbers of circulating B cells, hyper-IgM, and low proliferative responses induced by CD40 ligand. Lyn−/− mice also develop splenomegaly, produce autoreactive Abs with age, and finally develop glomerulonephritis. Another abnormality observed in lyn−/− mice is that their disability to form germinal centers (GCs). It has been considered that GCs play an important role in affinity maturation and differentiation to B cell memory upon immunization with thymus-dependent Ag. Since Lyn kinase has been thought to be downstream of the signals from the B cell Ag receptor as well as CD40, we studied whether or not lyn−/− mice could exhibit normal Ag-specific class switching and affinity maturation following somatic hypermutation. The mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin (NP-CG). Production of NP-specific IgG1 Abs was slightly reduced but clearly detectable. The affinity of Abs produced was comparable to that in wild-type mice. Furthermore, somatic hypermutation occurred in the heavy-chain variable region at the same level as that in wild-type mice. Therefore, we conclude that isotype switching and affinity maturation occur normally in lyn−/− mice without the formation of GCs. The results lead to a speculation that Lyn may not play a role in induction of isotype switching or affinity maturation, despite being downstream of the signals from the B cell Ag receptor complex and CD40, and that GC architecture may not be absolutely essential for affinity maturation.
Induction of Tolerance in B-1 Cells for Bromelain-Treated Mouse Red Blood Cells by a Transient Presence of Anti-Idiotype Antibodies in Neonatal and Adult Mice
Authors: Kawaguchi Susumu
Published: January 2023

Murine B-1 cells are thought to develop from Ig− progenitors early in ontogeny and to expand by self-renewal. To examine the early development of Ig+ precursors of B-1 cells for bromelain-treated mouse RBC, the transient presence of RidA, a rat anti-Id mAb for VH11/Vκ9-type anti-bromelain-treated mouse Abs, was produced in neonatal mice. The presence of RidA during days 0 to 10 of age resulted in an 80% reduction in peritoneal RidA-Id+ B cells and B cells secreting RidA-Id+ Ig after LPS stimulation in 8-wk-old mice. This suggests that most Ig+ precursors for adult RidA-Id+ B cells already exist in 10-d-old mice. However, RidA injected into mice on day 10 had to persist for >4 days to result in a significant reduction in adult B cells. Similarly, although RidA injected into adult mice bound immediately to membrane Ig (mIg) of the peritoneal RidA-Id+ B cells, a RidA persistence for >4 days was required to suppress LPS reactivity of peritoneal and splenic B cells. The binding of RidA to mIg preexisting on B cells has no apparent effect on the ability of neonatal B cells to expand clonally or on the ability of adult B cells to secrete RidA-Id+ Ig after LPS stimulation. Both abilities evidently are suppressed by the accumulation of reaction between freshly expressed mIg and RidA.
Respiratory Syncytial Virus-Infected Pulmonary Epithelial Cells Induce Eosinophil Degranulation by a CD18-Mediated Mechanism
Authors: Olszewska-Pazdrak Barbara , Pazdrak Konrad , Ogra Pearay L. , Garofalo Roberto P.
Published: January 2023

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the β2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.
Molecular Mechanisms of Inducible Nitric Oxide Synthase Gene Expression by IL-1β and cAMP in Rat Mesangial Cells
Authors: Eberhardt Wolfgang , Plüss Christoph , Hummel Richard , Pfeilschifter Josef
Published: January 2023

Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1β and cAMP predominantly at the transcriptional level. The 5′-flanking region of the rat iNOS gene contains several binding sites for transcription factors potentially involved in cytokine and cAMP signaling such as nuclear factor-κB/Rel, CCAAT/enhancer-binding protein (C/EBP), and cyclic AMP-responsive element-binding protein/ATF. We tested promoter activities of serial and site-directed deletion mutants of iNOS-chloramphenicol acetyltransferase reporter genes after transient transfection and stimulation of mesangial cells. A region between bp −277 and −111 bearing a CCAAT/enhancer-binding protein-response element was found to be critical for cAMP-mediated gene induction but dispensable for IL-1β inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to −111 containing a κB site is sufficient to confer IL-1β-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1β-inducible nuclear factor-κB p50/p65 heterodimeric complex. Using probes containing C/EBP-binding sites from the iNOS gene revealed further binding of different complexes, all of which were strongly inducible by cAMP and to a lower extent also by IL-1β. Abs against cyclic AMP-responsive element-binding protein, C/EBPβ, and C/EBPδ were able to partially supershift single complexes, suggesting the participation of these transcription factors in the regulation of iNOS gene expression by cAMP and IL-1β. Finally, we show that both cAMP and IL-1β strongly induce steady-state levels of C/EBPβ and C/EBPδ mRNA levels. These data demonstrate that IL-1β and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.
Posttranscriptional Lipopolysaccharide Regulation of the Lysozyme Gene at Processing of the Primary Transcript in Myelomonocytic HD11 Cells
Authors: Goethe Ralph , Phi-van Loc
Published: January 2023

Lysozyme is increasingly expressed in macrophages in inflammatory response to bacterial LPS. In this study, we investigated the mechanisms that control expression of the lysozyme gene in myelomonocytic HD11 cells activated by LPS. Nuclear run-on transcription assays showed that LPS caused a 15-fold increase in the transcription rate of the lysozyme gene. However, Northern analyses with lysozyme cDNA and intron sequences revealed that the LPS-induced increase in nuclear lysozyme transcripts greatly exceeded the increase in transcription rate. Furthermore, nuclear lysozyme transcripts in untreated cells with a t1/2 of <10 min were more unstable than those accumulated in LPS-activated cells. We suggested, therefore, that the increased lysozyme expression following LPS treatment was largely due to a nuclear stabilization of the primary transcript. Interestingly, the increase in stability of the lysozyme primary transcript was accompanied by changes in nuclear processing including an increase in poly(A) tail length, which gradually shortened after entering the cytoplasm. The long lysozyme poly(A) tail, however, did not result in any increase in polysomal recruitment for translation or in stability of the cytoplasmic lysozyme mRNA.