bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-15 functions as a potent autocrine regulator of macrophage proinflammatory cytokine production: evidence for differential receptor subunit utilization associated with stimulation or inhibition.
Authors: Alleva D G , Kaser S B , Monroy M A , Fenton M J , Beller D I
Published: December 2022

The cytokine IL-15 appears to mimic the stimulatory activity of IL-2 on lymphocytes by utilizing part of the IL-2R complex. Although effects of IL-15 on Mphi activities have not previously been reported, its derivation from activated Mphi suggested a possible autocrine role in regulating Mphi functions and prompted us to determine whether IL-15 modulated LPS-activated Mphi cytokine production. Whereas high IL-15 concentrations enhanced proinflammatory (i.e., TNF-alpha, IL-1, and IL-6) and anti-inflammatory (i.e., IL-10) cytokine production by two- to sixfold, extremely low IL-15 concentrations (picomolar to attomolar range) markedly and selectively suppressed Mphi proinflammatory, but not anti-inflammatory, cytokine production by two- to fourfold. The stimulation (but not the suppression) of TNF-alpha production by IL-15 required the (IL-2/IL-15) receptor beta chain, as demonstrated by receptor subunit-blocking studies and lack of stimulation of Mphi from IL-2Rbeta-deficient mice. Conversely, suppression most likely involved the alpha receptor (IL-15R alpha) because this high affinity receptor would be engaged by low concentrations of IL-15, and its inducible expression correlated with the degree of suppression in both a time- and LPS dose-dependent fashion. Moreover, Ab-mediated neutralization studies revealed that endogenous IL-15 activity regulated Mphi activation with kinetics similar to that seen in response to exogenously added IL-15: suppressor activity increased over time in correlation with IL-15R alpha gene expression. This study demonstrates a novel dose-dependent and autocrine activity of IL-15 in Mphi regulation.
IL-7 overexpression in transgenic mouse keratinocytes causes a lymphoproliferative skin disease dominated by intermediate TCR cells: evidence for a hierarchy in IL-7 responsiveness among cutaneous T cells.
Authors: Williams I R , Rawson E A , Manning L , Karaoli T , Rich B E , Kupper T S
Published: December 2022

IL-7 is a keratinocyte-derived lymphocyte growth factor critical for the development of gammadelta T cells including murine dendritic epidermal T cells (DETC). We derived transgenic mice that overexpress IL-7 in basal keratinocytes under the control of the human K14 promoter. These K14/IL-7 mice develop dermal and epidermal T cell infiltrates associated with alopecia. This lymphoproliferative skin disease is substantially more severe in mice homozygous for the K14/IL-7 transgene. Conventional DETC expressing a Vgamma5 Vdelta1 TCR are rare or absent among the cutaneous T cells in these mice. The T cells in the skin infiltrates of young K14/IL-7 mice are predominantly gammadelta T cells that express intermediate levels of TCR, are negative for E-cadherin, often lack expression of CD2, and include cells that coexpress NK1.1. T cells expressing intermediate levels of a TCR-alphabeta are also present in transgenic skin, and progressively increase in number as the mice age. Phenotypically similar intermediate gammadelta and alphabeta T cell subsets also constitute the major lymphocyte populations recovered from organ culture of normal mouse skin in the presence of IL-7, suggesting that the T cells that accumulate in the epidermis of K14/IL-7 mice are derived from precursors normally resident in skin. We conclude that intermediate TCR cells, some of which coexpress NK1.1, can be selectively expanded in skin under the influence of IL-7 produced locally. Our results also suggest that features of the epidermal microenvironment besides keratinocyte-derived IL-7 account for the normal predominance of Vgamma5 Vdelta1 DETC in mouse epidermis.
Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis.
Authors: Waldner H , Sobel R A , Howard E , Kuchroo V K
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease inducible in susceptible animals by myelin Ag-specific CD4+ Th1 cells. The mechanisms by which these cells induce inflammation and demyelination in the central nervous system (CNS) are incompletely understood. To determine the roles of Fas and FasL in the involvement of CNS autoimmune injury, we determined susceptibility to EAE of Fas-or FasL-deficient mice. Compared with wild-type mice, mice expressing lpr (Fas) and gld (FasL) mutations were relatively resistant to the development of clinical EAE, and this correlated with fewer inflammatory infiltrates and cells undergoing apoptosis in the CNS of the mutant mice. The gld and lpr mice, however, developed significant T cell responses with production of Th1 cytokines in response to the encephalitogenic myelin peptide. These results suggest that the Fas/FasL pathway plays a critical role in the development of EAE probably by mediating apoptosis within the target tissue.
Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization: implications for immunotherapy of prostate cancer.
Authors: Fong L , Ruegg C L , Brockstedt D , Engleman E G , Laus R
Published: December 2022

Prostatic acid phosphatase (PAP) is uniquely expressed in prostatic tissue and prostate cancer. In this study, the immunogenicity of PAP was investigated in a male rat model. We show that immunization with recombinant rat or human PAP in CFA leads to a significant Ab response, but does not generate CTL or result in autoimmune prostatitis. In contrast, immunization with recombinant vaccinia expressing human PAP, but not rat PAP, generates a CTL response and tissue-specific prostatitis in the absence of detectable PAP-specific Abs. These findings suggest that a cellular immune response to PAP, rather than Abs, mediates destructive autoimmune prostatitis. Thus, xenogeneic forms of PAP are a new tool for the induction of prostate-specific immunity and may prove useful for the immunotherapy of prostate cancer.
B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching.
Authors: Zelazowski P , Carrasco D , Rosas F R , Moorman M A , Bravo R , Snapper C M
Published: December 2022

The Ig heavy chain locus contains a number of binding sites for the transcriptional activator, c-Rel. In this study, we evaluated the capacity of B cells from mice made genetically deficient in the C-terminal, transactivation domain of the c-Rel protein (delta c-Rel) to undergo Ig class switching. Flow-cytometric and digestion circularization PCR analyses revealed that delta c-Rel B cells failed to switch to IgG3 in response to LPS alone, or to IgG1 or IgE in response to LPS + IL-4. This failure to switch to IgG3 or IgG1 was associated with a corresponding loss of germline CH gamma 3 or CH gamma 1 RNA. However, the defective switching to IgE in delta c-Rel B cells was associated with normal levels of germline CH epsilon RNA relative to control B cells. The ability of delta c-Rel B cells to switch to IgG1, in response to LPS + IL-4, could be restored through the action(s) of additional stimuli, and this was associated with induction of normal levels of germline CH gamma 1 RNA relative to controls. In contrast, LPS-activated B cells from delta c-Rel mice underwent normal switching to IgA in the presence of TGF-beta, relative to control B cells. This was associated with equivalent steady state levels of germline CH alpha RNA between the two B cell populations. These data are the first to demonstrate a key and selective role for c-Rel in the regulation of Ig class switching. Furthermore, distinct differences are revealed in the Ig isotype induction profiles of B cells lacking c-Rel activity vs those deficient in p50/nuclear factor-kappa B.
CTL generation in the presence of IL-4 is inhibited by free p40: evidence for early and late IL-12 function.
Authors: Abdi K , Herrmann S H
Published: December 2022

The cytokine requirements for the generation of a CTL response were examined using thymocyte responders stimulated with Con A. In agreement with previous reports, IL-4 alone can generate a strong CTL response. To determine whether IL-12 was required for this response, we added either a neutralizing Ab against IL-12 or an antagonist of IL-12 function, p40. Both anti-IL-12 and p40 were found to inhibit the generation of CTL in the presence of IL-4. Consistent with these data, exogenous IL-12 was found to synergize with IL-4, particularly, when IL-4 was added at levels unable to generate a CTL response alone. This synergistic response was observed whether IL-12 was added at the beginning of culture along with IL-4 or after the first day to cultures initiated with IL-4. Likewise, the combination of IL-2 plus IL-12 evoked a strong synergistic response when cultures were initiated with IL-2 and IL-12 was added initially or after the first day of culture. Addition of either IL-2 or IL-12 alone did not generate a response. In contrast to these data showing that IL-12 was able to signal late in culture, p40 inhibited CTL generation in response to IL-4 only when added at the beginning of culture. These results imply that CTL induction involves a crucial early IL-12 signal, which once delivered permits cells to respond to a later IL-12 signal. These data are consistent with the idea that free circulating p40 could inhibit the generation of CTL by antagonizing the early IL-12 "differentiation" signal.
Differential requirement of protein tyrosine kinases and protein kinase C in the regulation of T cell locomotion in three-dimensional collagen matrices.
Authors: Entschladen F , Niggemann B , Zänker K S , Friedl P
Published: December 2022

Locomotion of T lymphocytes within three-dimensional collagen matrices is regulated via different signaling states of the cells. Purified human CD4+ and CD8+ T cells developed a spontaneously locomoting subpopulation of about 25% of the whole population immediately after incorporation into a three-dimensional collagen matrix analyzed by time-lapse videomicroscopy. This spontaneous locomotion was accompanied by enhanced tyrosine phosphorylation of the focal adhesion kinase (FAK). Inhibition of protein tyrosine kinase (PTK) activity using genistein significantly reduced the spontaneous locomotory activity. This reduction was overcome by subsequent activation of protein kinase C (PKC) using PMA, which led to a persistent increase of locomotory activity to more than 60% of the cells. Thus, the PKC-driven type of locomotion was independent of PTK activity, whereas spontaneous locomotion was not altered by inhibition of PKC activity using calphostin C or inhibition of the serine/ threonine phosphatases pp1 and pp2A using okadaic acid. We presume that PTK activity, especially tyrosine phosphorylation of FAK, is decisively involved in the regulation of spontaneous T lymphocyte locomotion, which is independent of PKC activity. In contrast, PKC-driven locomotion is independent of tyrosine phosphorylation events, indicating that T lymphocyte locomotion is regulated by more than one signal transduction pathway. Furthermore, confocal microscopy analysis of phosphotyrosine residues, FAK, and PKC revealed an exclusive cellular distribution of these components, suggesting a regulation of T lymphocyte locomotion different from migration models developed for other cell types, which refer to a colocalization of FAK and PKC in focal adhesions.
The processing routes determined by negatively charged residues in DR1-restricted T cell determinants.
Authors: Robadey C , Ammerlaan W , Muller C , Cloutier I , Sékaly R P , Haefliger J A , Demotz S
Published: December 2022

The presentation pathways followed by DR1-restricted determinants from the fusion protein of measles virus were studied. By assessing the capacity of various APC preparations to stimulate fusion protein-specific T cells, it was shown that the determinant contained within the fusion protein sequence 254-268 (F254) relies on newly synthesized DR1 protein for its presentation. By contrast, the determinant contained within the fusion protein sequence 314-328 (F314) is captured by DR1 protein recycled from the plasma membrane. In vitro binding analyses showed that the F254 and F314 peptides optimally bind to DR1 at pH 4 and pH 5, respectively. In addition, it was found that binding of the F254 peptide to DR1 is much poorer at pH 7 than at pH 4, while binding of the F314 peptide was decreased only moderately at pH 7 as compared with pH 5. Substitution of the glutamic acid 261 for an alanine in the F254 peptide resulted in an analogue with an improved capacity of binding to DR1 at neutral pH. By contrast, replacement of the valine 319 by a glutamic acid in the F314 peptide generated an analogue with a decreased binding capacity at pH 7. These findings indicated that determinants that do not bear acidic residues are captured efficiently by DR1 molecules over a broader range of pH than determinants containing acidic residues. Binding analyses between DR1 and four additional peptides further supported this conclusion. Altogether, these results suggested that acidic residues, by tuning the optimal pH for the assembly of peptide-DR1 complexes, determine the processing pathway followed by the determinants.
Human endothelial cell costimulation of T cell IFN-gamma production.
Authors: Briscoe D M , Henault L E , Geehan C , Alexander S I , Lichtman A H
Published: December 2022

In this report, we show that human endothelial cells (EC) provide costimulatory signals to mitogen-activated CD4+ T cells to augment IFN-gamma production. We also show that EC can enhance responsiveness of the T cells to IL-12. While IL-12 has no effect on IFN-gamma production by cultured CD4+ T cells in the absence of EC, addition of IL-12 to T cell-EC cocultures augments IFN-gamma production by as much as fivefold. Separation of T cells from EC by a semipermeable membrane inhibits the effect of EC and IL-12 on IFN-gamma production. Anti-LFA-3 Abs, in the absence or presence of IL-12, inhibit EC costimulation of IFN-gamma production by up to 50%, while Abs to intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), LFA-1, and very late antigen-4 (VLA-4) have relatively little effect. Pretreatment of T cells with conditioned medium from T cell-EC cocultures, or with IL-2 or IL-1 alpha similarly primes CD4+ T cells for the costimulatory effect of IL-12 on IFN-gamma production. EC costimulation of IFN-gamma production is inhibited by cyclosporine. However, in the presence of IL-12 there is a marked resistance to this inhibitory effect, suggesting that the IL-12-induced costimulatory pathway is distinct from the costimulatory pathway activated by endothelium alone. Our data are consistent with the hypothesis that, as a consequence of interactions with endothelium, T cells that migrate into an inflammatory site are primed to have enhanced responses to Ag and cytokine(s), such as IL-12, that influence T cell-cytokine production.
Identification of a novel MHC class I gene, Mamu-AG, expressed in the placenta of a primate with an inactivated G locus.
Authors: Boyson J E , Iwanaga K K , Golos T G , Watkins D I
Published: December 2022

Maternal tolerance of the fetal allograft remains poorly understood. In humans, expression of the highly polymorphic classical HLA-A and HLA-B loci is suppressed, while expression of the nonclassical HLA-G locus is up-regulated at the maternal-fetal interface. Like other nonclassical MHC class I molecules, HLA-G exhibits limited diversity, but certain characteristics of HLA-G distinguish it from other nonclassical MHC class I molecules: it has a truncated cytoplasmic domain, it is the product of alternatively spliced mRNAs, and it is expressed primarily in the placenta. We have examined MHC class I expression in the placenta of the rhesus monkey to determine whether this animal is a suitable model in which to study the function of HLA-G. Although the rhesus monkey possesses orthologs of many MHC class I and II loci found in humans, the HLA-G ortholog is a pseudogene in this nonhuman primate species. In this study, we report the identification of a novel nonclassical MHC class I locus expressed in the placenta of the rhesus monkey, Mamu-AG (Macaca mulatta-AG). Although unrelated to HLA-G, Mamu-AG encodes glycoproteins with all of the characteristics of HLA-G. These Mamu-AG glycoproteins are limited in their diversity, possess truncated cytoplasmic domains, are the products of alternatively spliced mRNAs, and their expression is restricted to the placenta. Taken together, these data suggest that convergent evolution may have resulted in the expression of a unique nonclassical MHC class I molecule in the rhesus monkey placenta, and that the common structural features of Mamu-AG and HLA-G may be functionally significant.
Domain-switched mouse IgM/IgG2b hybrids indicate individual roles for C mu 2, C mu 3, and C mu 4 domains in the regulation of the interaction of IgM with complement C1q.
Authors: Chen F H , Arya S K , Rinfret A , Isenman D E , Shulman M J , Painter R H
Published: December 2022

Although polymeric IgM and monomeric IgG are potent activators of the classical complement pathway, previous studies have indicated that monomeric IgM is inactive. To understand this and to examine the roles of the individual mu domains in complement activation, we created a set of IgM/IgG2b mouse chimeric Abs in which homologous domains of both Abs have been interchanged, either singly or together with adjacent domains. The monomer subunits (H2L2) of the resulting chimeras were analyzed for their capacities to bind C1q and to initiate complement-mediated lysis (CML) of haptenated erythrocytes. When C gamma 2 was flanked by C mu 4, the inherent C1q-binding activity of the C gamma 2 domain was lost. This demonstrates that C mu 4 can suppress the C1q-binding activity of the adjacent C gamma 2 domain, and suggests that C mu 4 may exert a similar effect on the C mu 3 domain in the IgM monomer subunit. When C mu 3 was located in an IgG2b background and potentially freed from the constraints imposed by the IgM background, the monomer was not able to bind C1q or initiate CML. This suggests that these activities are not expressed inherently in the C mu 3 domain. The transplantation of C mu 3 together with C mu 4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although neither the monomer nor the polymer forms were active in CML; conversely, all IgM polymers with a transplanted C gamma 2 domain were active in both C1q binding and CML, and demonstrated apparent Kd values similar to that of wild-type IgM.
Flexibility of human IgG subclasses.
Authors: Roux K H , Strelets L , Michaelsen T E
Published: December 2022

A variable region (Id)-matched set of genetically engineered human IgG1, -2, -3, and -4 subclass molecules was analyzed by electron microscopy for hinge-mediated differences in flexibility. The hinge-mediated bending was studied, as was the ability of the subclasses to form immune complexes with two anti-Id mAbs. The data show that the rank order (most to least flexible) of the IgG subclasses for hinge-folding mode of flexibility between Fab arms is IgG3 > IgG1 > IgG4 > IgG2. The mean Fab-Fab angles for the subclasses are IgG3, 136 degrees; IgG4, 128 degrees; IgG2, 127 degrees; and IgG1, 117 degrees. Fab-Fc angles were similarly analyzed. By sampling of equimolar mixtures of Id-bearing IgGs and each of two anti-Id mAb after incubation over time (1.5 min to 3.5 h), different kinetic profiles of immune complex formation of defined geometry were documented. Both anti-Id mAbs displayed unique kinetic profiles when complexed with the four IgG subclass molecules but also shared important features. Most notable was the higher propensity to form closed bivalent ring Id-anti-Id dimers with IgG3 than with IgG2 and IgG4. IgG1 was intermediate in its ability to form such dimers.
TNF initiates E-selectin transcription in human endothelial cells through parallel TRAF-NF-kappa B and TRAF-RAC/CDC42-JNK-c-Jun/ATF2 pathways.
Authors: Min W , Pober J S
Published: December 2022

TNF acts on the E-selectin gene promoter at three kappa B elements and at a variant cAMP-responsive element that binds ATF2/c-Jun. In human endothelial cells, TNF rapidly induces N-terminal domain phosphorylation of both c-Jun and ATF2. Transient overexpression of N-terminal truncated c-Jun or catalytically inactive Jun N-terminal kinase (JNK) 1 and 2 inhibits TNF-induced transcription of an E-selectin but not a kappa B promoter-reporter gene. Transient overexpression of the TRAF2 adaptor protein can activate NF-kappaB and endogenous JNK, whereas N-terminal truncated TRAF2 protein blocks TNF-induced NF-kappa B and JNK activation as well as E-selectin promoter-reporter gene transcription. Transient overexpression of RAC1 or CDC42, but not RAS, constitutively activates JNK and augments TNF-induced E-selectin transcription. Finally, transient overexpression of catalytically inactive JNK or truncated TRAF2 partially inhibits endogenous E-selectin protein expression in human endothelial cells. These data suggest that TNF activates parallel TRAF-NF-kappa B and TRAF-RAC/CDC42-JNK-c-Jun/ATF2 pathways to initiate E-selectin transcription.
Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions.
Authors: Pollack M , Ohl C A , Golenbock D T , Di Padova F , Wahl L M , Koles N L , Guelde G , Monks B G
Published: December 2022

Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors.
Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.
Authors: Panesar M , Papillon J , McTavish A J , Cybulsky A V
Published: December 2022

In rat membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. In cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of phospholipase A2 (PLA2). To address mechanisms of PLA2 activation, GEC were stably transfected with cDNAs of wild-type cytosolic PLA2 (cPLA2-wt), or group II secretory PLA2, producing overexpression of PLA2 activity. Sublytic C5b-9 markedly increased free [3H]AA in cPLA2-wt-transfected GEC, but only trivial increases were evident in secretory PLA2-transfected, or neo (control) GEC. In cPLA2-wt-transfected GEC, reduction of extracellular free Ca2+ or down-regulation of protein kinase C inhibited [3H]AA release. To further address the regulation of cPLA2, we stably expressed a mutant cPLA2 in which the Ca2+-dependent lipid binding domain was deleted (deltaCaLB). In GEC that express cPLA2-deltaCaLB, the C5b-9-induced increase in free [3H]AA was comparable with neo, despite expression of cPLA2-deltaCaLB at levels similar to cPLA2-wt. We then stably expressed another cPLA2 mutant (cPLA2-srcmyr) in which the CaLB domain was replaced by the N-terminal myristoylation domain of c-Src. cPLA2-srcmyr is permanently membrane associated. At low extracellular free Ca2+, C5b-9 increased free [3H]AA significantly in GEC that express cPLA2-srcmyr, while in neo GEC, the change was negligible. Thus, C5b-9 activates the cPLA2 isoform. Activation is dependent on the CaLB domain, and is mediated by phosphorylation, Ca2+ influx, and membrane association.
Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-alpha.
Authors: Tessier P A , Naccache P H , Clark-Lewis I , Gladue R P , Neote K S , McColl S R
Published: December 2022

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.
Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis.
Authors: Ghivizzani S C , Kang R , Georgescu H I , Lechman E R , Jaffurs D , Engle J M , Watkins S C , Tindal M H , Suchanek M K , McKenzie L R , Evans C H , Robbins P D
Published: December 2022

To investigate the pathophysiologic effects of chronically elevated intra-articular levels of IL-1 beta, we used an ex vivo gene transfer method to deliver and express human IL-1 beta (hIL-1 beta) in the knee joints of rabbits. Expression of hIL-1 beta resulted in a severe, highly aggressive form of arthritis analogous to chronic rheumatoid arthritis in humans. Intra-articular manifestations included intense inflammation, leukocytosis, synovial hypertrophy and hyperplasia, and highly aggressive pannus formation with erosion of the articular cartilage and periarticular bone. Systemic effects were also observed, including diarrhea, fever, weight loss, and an increased erythrocyte sedimentation rate. In addition, the hIL-1 beta was found to induce elevated levels of both rabbit IL-1 beta and TNF-alpha in synovial fluid. Following the loss of hIL-1 beta transgene expression between 14 and 28 days post-transplantation, many of these changes began to normalize. These results suggest that chronically elevated intra-articular levels of IL-1 beta alone are sufficient to produce virtually all the pathologies found in rheumatoid arthritis, and furthermore, demonstrate that gene transfer can be used to investigate the roles of specific gene products in the pathogenesis of arthritis.
A synthetic peptide administered with IL-12 elicits immunity to Listeria monocytogenes.
Authors: Miller M A , Skeen M J , Ziegler H K
Published: December 2022

IL-12 is a pivotal cytokine signal for the development of Th1-type cellular responses that are required for control of intracellular pathogens. We previously demonstrated that coinjection of IL-12 with heat-killed Listeria monocytogenes, which was not immunogenic when injected alone, elicited intense Ag-specific T cell responses that conferred protection against subsequent challenge with Listeria. Herein we describe the remarkable finding that a nonimmunogenic synthetic peptide corresponding to a dominant MHC class II (H-2k)-restricted listerial determinant, when coinjected i.p. with murine IL-12, elicited potent Ag-specific immune responses that conferred protective immunity against Listeria.
In vitro and in vivo evidence for high frequency of I-Ab-reactive CD4+ T cells in HLA-DQ or HLA-DRA transgenic mice lacking endogenous MHC class I and/or class II expression.
Authors: Ishimoto T , Yamamoto K , Fukui Y , Fukuda Y , Dohi K , Sasazuki T
Published: December 2022

Although T cells are educated to recognize foreign antigenic peptides in the context of self MHC molecules during their development in the thymus, peripheral T cells also recognize allo- and xeno-MHC molecules. The lower frequency of xeno-MHC-reactive T cells than that of allo-MHC-reactive T cells is often explained by the difference in the degree of homology between xeno- or allo-MHC and self MHC molecules, as well as by the species barrier of the molecules involved in immune recognition. To distinguish these two possibilities, we estimated the frequency of I-Ab-reactive CD4+ T cells selected by HLA-DQ or DR alpha E beta b molecules, using HLA-DQ6 and HLA-DRA transgenic C57BL/6 (B6) mice lacking endogenous MHC class I and/or class II molecules (DQ6A0/0 and DR alpha 30A0/0 beta 20/0). CD4+ lymph node T cells from DQ6A0/0 and DR alpha 30A0/0 beta 20/0 showed the strong proliferative response to I-Ab molecules. In addition, DQ6A0/0 and DR alpha 30A0/0 beta 20/0 rejected the skin graft from mice expressing I-Ab molecules irrespective of MHC class I expression, indicating that the CD4+ T cells recognizing I-Ab molecules are directly involved in this rejection. The estimated frequency of I-Ab-reactive CD4(+)CD8- thymocytes in DR alpha 30A0/0 beta 20/0 and DQ6A0/0 was comparable with that observed in the MHC class II-disparate strains. Our findings thus indicate that CD4+ T cells selected to mature on xeno-MHC class II molecules such as HLA-DQ6 or DR alpha E beta b, when these molecules are expressed in mice, recognize I-Ab molecules as allo-MHC class II, despite the less structural homology.
Costimulatory signals through B7.1/CD28 prevent T cell apoptosis during target cell lysis.
Authors: Daniel P T , Kroidl A , Cayeux S , Bargou R , Blankenstein T , Dörken B
Published: December 2022

Expression of B7 on tumor cells can circumvent T cell tolerance and lead to the generation of tumor cell-specific T cell immunity. The effect of B7 expression on the generation of protective antitumor immunity has been attributed primarily to 1) more efficient T cell activation and 2) better generation of tumor-specific killer T cells. We have investigated the role of costimulation through B7.1 and its receptor, the CD28 molecule, in the generation of allogeneic human CTLs against MCF-7 breast cancer cells. In this setting, we describe how activated CTLs undergo activation-induced cell death upon killing the target cell. Instead of proliferation and clonal expansion, the majority of the CTLs underwent apoptotic cell death. CTL apoptosis could be blocked by 50% when binding of the Fas ligand to its receptor, the CD95 (APO-1/Fas) molecule, was prevented. Fas ligand was detected in the activated T cells, but not in MCF-7 or a panel of other breast cancer cell lines. This excludes an active role for MCF-7 during CTL death and indicates that the CTL apoptosis is due to an autocrine production of the Fas ligand by CTLs. Costimulation of CTLs by retrovirally B7.1-transfected MCF-7 drastically reduced the sensitivity of the CTLs to apoptosis during target contact. Thus, in tumor cell vaccination, B7.1 might play a major role in preventing T cell death by altering T cell susceptibility for apoptosis.
Human CD38, a leukocyte receptor and ectoenzyme, is a member of a novel eukaryotic gene family of nicotinamide adenine dinucleotide+-converting enzymes: extensive structural homology with the genes for murine bone marrow stromal cell antigen 1 and aplysian ADP-ribosyl cyclase.
Authors: Ferrero E , Malavasi F
Published: December 2022

The human leukocyte Ag CD38 is a 45-kDa type II membrane glycoprotein that functions both as a transmembrane signaling receptor and as an ectoenzyme. CD38 can transmit positive or negative signals regulating T and B lymphocyte proliferation and differentiation and can enzymatically convert nicotinamide adenine dinucleotide to cyclic ADP-ribose, a potent intracellular calcium releaser. To begin the genetic analysis of CD38, we have isolated and characterized the human CD38 gene, previously mapped to chromosome 4. Human CD38 is encoded by a single copy gene that extends over > 62 kb and consists of eight exons and seven introns, including a very long intron that interrupts the 5' coding region. The 5' upstream region of the gene is characterized by the absence of canonical TATA and CAAT boxes, the presence of a GC-rich tract immediately upstream of the initiator codon, several transcription start sites, and potential binding sites for the immunologic transcription factors T cell-specific transcription factor alpha, nuclear factor-IL-6, and interferon responsive factor-1. Comparison of the genomic structure of human CD38 with that of the genes for murine bone marrow stromal Ag bone marrow stromal cell antigen 1/BP-3 and aplysian ADP-ribosyl cyclase, which encode proteins related to CD38 in sequence and function, reveals their striking structural similarity. This strongly suggests that the three genes evolved from a common ancestor from which CD38 and bone marrow stromal cell antigen 1 arose by gene duplication before the divergence of humans and rodents.
Autoantibodies in mice lacking terminal deoxynucleotidyl transferase: evidence for a role of N region addition in the polyreactivity and in the affinities of anti-DNA antibodies.
Authors: Weller S , Conde C , Knapp A M , Levallois H , Gilfillan S , Pasquali J L , Martin T
Published: December 2022

The generation of terminal deoxynucleotidyl transferase knockout mice (TdT0) has demonstrated that TdT is the only major activity involved in N region addition. This enzyme generates diversity by adding random nucleotides at the V-D-J junctions and by disrupting the formation of repetitive "homology-directed" junctions. Several studies have demonstrated that the Ig heavy chain third complementarity-determining region (H-CDR3) and the N region play a critical role: 1) in distinguishing between polyreactive and monospecific combining sites in natural and Ag-induced Abs; and 2) in the specificity and polyreactivity of natural autoantibodies (autoAbs) and in particular of anti-DNA Abs. To examine the impact of the lack of TdT on the natural autoAb repertoire in adult mice, we have stimulated TdT0 and TdT+ littermates with LPS. Serum studies demonstrate that TdT is not critical for the generation of B cells expressing autoAbs including anti-DNA Abs and rheumatoid factors. However, the generation of a large collection of hybridomas indicates that the frequencies of these cells are reduced in TdT0 mice mainly due to a lower incidence of polyreactivity; also, the lack of N region diversity seems to negatively affect the affinity of anti-DNA Abs. The physiologic relevance of these data is discussed.
Cross-linking of Fc gamma receptor IIa and Fc gamma receptor IIIb induces different proadhesive phenotypes on human neutrophils.
Authors: Kocher M , Siegel M E , Edberg J C , Kimberly R P
Published: December 2022

Activation of polymorphonuclear leukocytes (PMN) plays an important role in vascular injury associated with systemic vasculitis and in models of autoantibody- and immune complex-mediated disease. The potential role of intravascular activation of PMN, however, is confounded by the observation that some stimuli injected i.v. (e.g., IL-8 and C5a) lead to L-selectin shedding by PMN, which inhibits attachment to endothelium and may be functionally anti-inflammatory. To explore the impact of Fc gamma receptor (Fc gamma R)-mediated activation on the PMN adhesive phenotype, Fc gamma RIIa (CD32) and Fc gamma RIIIb (Cd16) were targeted with receptor-specific reagents, and the expression of adhesion molecules-mediating rolling (L-selectin) and firm adhesion (CD11b/CD18) was measured. Engagement of either Fc gamma RIIa or Fc gamma RIIIb leads to activation, demonstrated by degranulation (upregulation of CD66b), and to increased expression of total CD11b/CD18 and functional CD11b/CD18 (I-domain). In contrast, L-selectin shedding induced by PMN Fc gamma R was divergent. Despite the 5- to 10-fold greater expression and engagement at saturation, activation via Fc gamma RIIIb led to little or no change in L-selectin expression. Stimulation of PMN with intact murine anti-receptor IgG1 showed a contribution of Fc gamma RIIa receptor polymorphisms, underscoring the direct influences of Fc gamma R allotypes on receptor function. These observations suggest that Fc gamma RIIIb-mediated activation of circulating PMN may lead to a proadhesive phenotype likely to promote systemic vascular damage. This Fc gamma R-mediated adhesive phenotype will vary with the receptors engaged and their allotypes, which, in turn, reflect properties of the immune complex and the genetics of the host.
IFN-gamma inhibits double-stranded RNA-induced E-selectin expression in human endothelial cells.
Authors: Faruqi T R , DiCorleto P E
Published: December 2022

IFN-gamma plays a role in immune regulatory functions as well as in viral defense. We show in this study that IFN-gamma treatment down-regulates the induction by a viral mimetic, polyinosinic-polycytidylic acid (poly(I:C)), of the endothelial cell-specific leukocyte adhesion protein, E-selectin. The inhibitory effect of IFN-gamma on poly(I:C)-induced E-selectin was concentration and time dependent and was specific for dsRNA, in that the induction of E-selectin by TNF-alpha, IL-1 beta, thrombin, or LPS was not inhibited significantly by this pretreatment. IFN-gamma pretreatment reduced poly(I:C)-induced E-selectin mRNA in a protein synthesis-independent manner. Poly(I:C)-induced E-selectin mRNA t1/2 was reduced slightly by IFN-gamma treatment, while the message for VCAM-1 was stabilized. Transient transfection of endothelial cells with an E-selectin promoter-driven reporter gene construct revealed that poly(I:C) stimulation of E-selectin promoter activity was decreased significantly by IFN-gamma pretreatment. Poly(I:C)-induced nuclear factor-kappa B activation following IFN-gamma pretreatment was unaffected, as shown by electrophoretic mobility shift analysis. These results indicate a novel role for IFN-gamma in the regulation of E-selectin gene expression in response to dsRNA by a transcriptional mechanism independent of nuclear factor-kappa B, as well as by a minor decrease in message stability.
Erythromycin A-derived macrolides modify the functional activities of human neutrophils by altering the phospholipase D-phosphatidate phosphohydrolase transduction pathway: L-cladinose is involved both in alterations of neutrophil functions and modulation of this transductional pathway.
Authors: Abdelghaffar H , Vazifeh D , Labro M T
Published: December 2022

All erythromycin A derivatives, irrespective of the size of the lactone ring and the nature of the substituent, inhibit oxidant production by neutrophils and promote their degranulation. We demonstrate in this study that the L-cladinose at position 3 of the lactone ring is a key structure in the modulation of these two neutrophil functions, suggesting that this sugar (alone or combined with a lactone structure) interferes with cell target(s) involved in both oxidant production and exocytosis. Taking roxithromycin as an example of erythromycin A derivatives, we also found that these molecules interfered with the phospholipase D (PLD)-phosphatidate phosphohydrolase pathway in two ways. In nonstimulated neutrophils, roxithromycin and all L-cladinose-bearing molecules activated PLD, as reflected by 1-O-[3H]alkyl-2-acyl-phosphatidyl-ethanol production. In addition, these drugs induced an accumulation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA), but not 1-O-[3H]alkyl-2-acylglycerol. PA accumulation seems to be involved in the induction of exocytosis by macrolides, as the roxithromycin-induced release of granular enzymes was impaired strongly in the presence of ethanol. By contrast, in stimulated neutrophils, roxithromycin inhibited PLD activity and totally impaired 1-O-[3H]alkyl-2-acylglycerol production. The inhibition of diglyceride production by roxithromycin (not its descladinosyl derivative) could explain its inhibitory effect on oxidant production. The relevance of our data to the clinical situation, particularly the anti-inflammatory activity of these drugs, requires further investigation.
Bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model.
Authors: Taylor R P , Martin E N , Reinagel M L , Nardin A , Craig M , Choice Q , Schlimgen R , Greenbaum S , Incardona N L , Ochs H D
Published: December 2022

We used Anger camera imaging in a monkey model to investigate the organ localization of a prototype particulate pathogen, 131I-labeled bacteriophage phi X174, after it was bound to the primate erythrocyte complement receptor and then cleared from the circulation. This 131I-labeled phi X174 was infused into the circulation of an immunized monkey, and the nascently formed immune complexes showed rapid and quantitative binding to erythrocytes via the immune adherence reaction (complement-mediated binding). Alternatively, phi X174 was infused into the circulation of a naive animal, and then cross-linked bispecific mAb complexes (heteropolymers, anti-CR1 x anti-phi X174) were infused into the circulation. The infused heteropolymers also facilitated rapid and quantitative binding of phi X174 to erythrocytes. In both cases, after a short lag period, the erythrocyte-bound phi X174 was rapidly cleared from the circulation, and the vast majority of the radiolabel was cleared to the liver, with a small amount clearing to the spleen. Further liver imaging confirmed that within 24 h most of the bacteriophage previously cleared to the liver via the heteropolymer system was phagocytosed and destroyed. The findings in this model system provide additional evidence for the potential utility of heteropolymers to facilitate the safe and rapid clearance of blood-borne pathogens as a potential treatment for infectious diseases.
The ligand binding site of the formyl peptide receptor maps in the transmembrane region.
Authors: Miettinen H M , Mills J S , Gripentrog J M , Dratz E A , Granger B L , Jesaitis A J
Published: December 2022

We propose that the N-formyl-Met-Leu-Phe binding site in the human neutrophil formyl peptide receptor (FPR) lies in the predicted transmembrane region. We examined the expression, binding, and G protein coupling of 28 mutated forms of FPR in stably transfected Chinese hamster ovary cells. The amino acids we mutated are: 1) predicted to be oriented toward the interhelical space; 2) analogous to those required for ligand binding in various other G protein-coupled receptors; 3) divergent from lipoxin A4 receptor, a low affinity receptor for formylated peptides; and 4) either highly conserved or divergent in other G protein-coupled receptors. Some mutations resulted in intracellular retention, suggesting that the receptors were misfolded. Most mutated receptors that were transported to the plasmalemma bound f-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with affinities similar to the wild-type receptor (Kd = 6 nM). However, mutations L78A (helix II), D106N, L109A (helix III), T157A (helix IV), R201A, I204Y, and R205A (helix V), W254A and Y257A (helix VI), and F291A (helix VII) resulted in reduced affinities (Kd = 30-128 nM). Of these mutations, D106N, R201A, and R205A also appeared to affect G protein coupling, suggesting that these residues may also be involved in signal transduction and/or are essential for proper folding of the molecule. Some of the FPR residues that appeared to be involved in binding of formylated peptides were located at sites analogous to those identified in ligand binding to certain other G protein-coupled receptors. It is thus possible that several G protein-coupled receptors have a common placement of ligand-binding amino acids.
Studies on the Antigenic Substances of CL. Botulinum XXXIII
Authors: Lommel Jeanne , Gunnison Janet B.
Published: December 2022

Although many studies on the antigenic complex of the bacterial cell have been made, the nature of the specific substances of an anaerobic organism has, as far as we know, not been investigated. Cl. botulinum was chosen since the species is divided into serologic groups as well as into toxicologic types. This work is an attempt to determine the nature of the substances responsible for the specificity. A modification of the alcoholic extraction method employed by Landsteiner and Levine (1) in their recent study on cholera vibrio was used for preparation of the antigenic reagents.

Experimental Procedure. The extracts were made as follows: The organisms grown for eighteen hours at 37°C. were centrifuged from 20 liters of casein digest broth, washed twice in ice cold saline to prevent autolysis; then placed in 500 cc. of 75 per cent alcohol and allowed to stand at room temperature for three days.
Differential Effect of Anti-B7-1 and Anti-M150 Antibodies in Restricting the Delivery of Costimulatory Signals from B Cells and Macrophages
Authors: Agrewala Javed N. , Suvas Susmit , Verma Rakesh K. , Mishra Gyan C.
Published: December 2022

B7-1 and M150 are potent costimulatory molecules expressed on B cells and macrophages. We have examined the capacity of Abs against B7-1 and M150 in differentially inhibiting the costimulatory signals delivered by macrophages and B cells to OVA-specific CD4+ T cells. The anti-B7-1 Ab significantly blocked the proliferation of Th cells, MLR, T cell help to B cells, and secretion of IFN-γ when B cells were used to provide costimulation, but not when macrophages were used. In contrast, anti-M150 Ab significantly decreased the proliferation of Th cells, MLR, and production of IFN-γ, when macrophages were utilized to provide costimulatory signals, but not when B cells were used as APC. However, when macrophages activated with IFN-γ were used as a source of costimulation, like anti-M150 Ab, Ab to B7-1 also down-regulated the activation of Th cells. The significance of this finding is that M150 is a potent first costimulatory signal for initiating proliferation and secretion of IFN-γ and providing cognate help for B cells by Th cells when the macrophage is used as an accessory cell. M150-induced IFN-γ production induces the expression of B7-1 on the surface of macrophages, which then delivers a second cosignal for Th cells. B7-1 works efficiently when B cell provides cosignal. Both of the molecules promote Th1 activity, as evidenced by the inhibition of the secretion of IFN-γ but not IL-4 by Th cells with anti-M150 and B7-1 Abs.