bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of a hot spot for DNA insertion suggests a mechanism for Ig switch recombination.
Authors: Baar J , Pennell N M , Shulman M J
Published: January 2023

We recently reported that transfected DNA inserts into the VDJ-Cmu intron much more frequently than into average DNA, and that insertion within this intron occurs preferentially into the switch region. To gain information about the mechanisms involved in DNA insertion, we sequenced the 5' and 3' junctions of typical transformants. Although the junction sequences did not indicate a preferred insertion motif within the switch region, our results suggest that joining of the transfected and chromosomal DNAs is facilitated by short regions of identity. Our analysis of the insertions into the non-switch part of the intron suggests that breakage of the chromosomal DNA occurs preferentially at sites that are flanked by short complementary sequences. This correlation suggests that the self-complementary DNA might form short stem-loops, which, in turn, are prone to enzymatic cleavage and thus facilitate the insertion of transfected DNA. A model is proposed in which this effect can account for both the higher than average frequency of insertion into the VDJ-Cmu intron and the preference for the switch region within this intron. An extension of this model is proposed to explain why the repetitive switch regions are the preferred breakage/rejoining sites for isotype switch rearrangements.
Multiple types of chimeric germ-line Ig heavy chain transcripts in human B cells: evidence for trans-splicing of human Ig RNA.
Authors: Fujieda S , Lin Y Q , Saxon A , Zhang K
Published: January 2023

Germ-line transcripts from Ig heavy chain loci precede the occurrence of isotype switching and are thought to play an important though still controversial role in Ig class switching. In this study, we employed a reverse transcriptase-PCR approach to detect human chimeric Ig germ-line mRNA transcripts. Multiple types of chimeric Ig germ-line transcripts (Imu-Cepsilon, Iepsilon-Cmu, Imu-Cgamma4, Igamma-Cmu, Igamma-Cepsilon, Iepsilon-Cgamma, and Igamma4-Calpha1 transcripts) were readily detected in human B cells stimulated with IL-4 alone. Sequence analysis revealed that all of these chimeric Ig germ-line transcripts represented the I exons from one Ig locus spliced to the CH exons from another locus by using consensus sequences for splicing donor and acceptor sites, indicating that they were generated through splicing machinery. In the case of stimulation of human resting B cells with IL-4 alone, the chimeric Ig germ-line transcripts are likely derived from a trans-splicing mechanism, as the extensive searching did not find evidence that Ig class-switch recombination had occurred, which alternatively could give rise to chimeric Ig mRNA by mechanisms other than trans-splicing. Similarly, an EBV-transformed gamma2 rearranged B cell line, GM1500, which produces IgG2 and contains both gamma2 productive and epsilon germ-line transcripts, also expressed chimeric germ-line RNA (Iepsilon-Cgamma2) and epsilon-productive transcripts (VDJ-Cepsilon). This line had no further sequential Sgamma2-Sepsilon rearrangements, providing evidence that the productive VDJ-Cepsilon mRNA was derived from a transcriptionally active unrearranged epsilon gene locus by trans-splicing. Taken together, these results provide possible evidence that trans-splicing of germ-line Ig RNA transcripts occurs in human B cells.
A critical role of Sp1- and Ets-related transcription factors in maintaining CTL-specific expression of the mouse perforin gene.
Authors: Youn B S , Kim K K , Kwon B S
Published: January 2023

This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin.
Control of parasitemia and survival during Trypanosoma brucei brucei infection is related to strain-dependent ability to produce IL-4.
Authors: Bakhiet M , Jansson L , Büscher P , Holmdahl R , Kristensson K , Olsson T
Published: January 2023

We studied non-MHC gene-dependent expression of a number of cytokines in relation to host defense and survival during Trypanosoma brucei brucei (Tbb) infection in mice. In particular, the role of IL-4 was explored with use of genomically IL-4-disrupted mice and in vivo Ab blocking. Splenocytes from MHC-identical B10.Q (relatively resistant) mice showed day 5 postinfection higher numbers of IL-4 mRNA expressing cells than C3H.Q (highly susceptible). A trypanosome-derived lymphocyte triggering factor, which is released by Tbb to polyclonally activate CD8+ T cells, stimulated naive splenocytes in vitro to a higher IL-4 response in B10.Q than in C3H.Q mice. The C3H.Q mice developed an extremely high parasitemia, showed a low Ab response against the variant surface glycoprotein (VSG), and had a mean survival time of 42 days. Conversely, B10.Q mice had lower parasitemia, mounted higher anti-VSG response, and had a mean survival time of 56 days. Deletion of the IL-4 gene had no influence on the infection in C3H.Q mice, while in B10.Q mice the deletion was associated with lower anti-VSG Ab levels and higher parasitemia. Paradoxically, B10.Q mice with disrupted IL-4 gene survived longer than the wild type. Anti-IL-4 Ab-blocking experiments in vivo displayed an enhanced parasitemia and prolonged survival in infected B10.Q mice. We conclude that 1) a non-MHC gene-related and CD8+-dependent ability to produce IL-4 partly determines the susceptibility to Tbb infection; and 2) IL-4, although involved in controlling the levels of parasitemia by its effects on immunoglobulin synthesis, also can have toxic effects on the animals.
Induction of nitric oxide synthase in mouse dendritic cells by IFN-gamma, endotoxin, and interaction with allogeneic T cells: nitric oxide production is associated with dendritic cell apoptosis.
Authors: Lu L , Bonham C A , Chambers F G , Watkins S C , Hoffman R A , Simmons R L , Thomson A W
Published: January 2023

Nitric oxide (NO) is an important effector molecule that is involved in immune regulation and host defense. In this study, highly purified NLDC 145+ (DEC-205+) MHC class II(bright) B7-2+ dendritic cells (DC) propagated from normal mouse bone marrow in response to granulocyte-macrophage CSF + IL-4 were induced to produce NO by IFN-gamma and LPS. NO production was inhibited by the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (NMMA). Nitrite also accumulated in mixed leukocyte culture supernatants as the result of coculture of DC with purified naive allogeneic T cells. Furthermore, NO production was induced by CD40 ligation. Suboptimal T cell proliferation observed at high relative concentrations of DC correlated with increased NO production and was mitigated by NMMA. Induction of mRNA for an inducible NOS (iNOS) in DC was confirmed by Northern blotting, whereas intracellular iNOS was visualized by two-color flow cytometry and by both immunofluorescent and immunogold labeling in a subpopulation of IFN-gamma + LPS-stimulated cells. Both endogenous NO production and exposure of unstimulated DC to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) resulted in DC apoptosis. Thus, although DC function initially as the most potent APCs for T cell activation, DC induced to synthesize NOS by IFN-gamma may inhibit (allogeneic) T cell proliferation: NO may suppress lymphocyte proliferation and also induce apoptosis of the most potent source of alloantigenic stimulation.
CD63 associates with tyrosine kinase activity and CD11/CD18, and transmits an activation signal in neutrophils.
Authors: Skubitz K M , Campbell K D , Iida J , Skubitz A P
Published: January 2023

As a member of the tetraspan family, it has been hypothesized that CD63 may be associated with signal transduction; however, its role in leukocyte function is unknown. To examine the potential ability of CD63 to activate neutrophils, the effects of five CD63 mAbs, AHN-16, -16.1, -16.2, -16.3, and -16.5, were examined for their ability to alter neutrophil adhesion to HUVEC monolayers. These CD63 Abs increased neutrophil adhesion to resting and TNF-stimulated HUVEC monolayers. This increase in neutrophil adhesion caused by CD63 Abs was blocked by a CD18 Ab and was associated with up-regulation of CD11/CD18 and down-regulation of CD62L on the neutrophil surface. CD11/CD18 was also found to be associated with CD63. This increase in neutrophil adhesion required physiologic extracellular calcium concentrations at or near the time of CD63 Ab binding. The incubation of CD63 Abs with cells in the absence of calcium for 10 min before repletion of calcium resulted in no increase in neutrophil adhesion. Protein kinase activity was detected in neutrophils associated with CD63. Most of the protein kinase activity associated with these Ags was tyrosine kinase activity, with a lesser amount of threonine and serine kinase activities. Src family kinases Lyn and Hck accounted for much of the associated tyrosine kinase activity. The data suggest that CD63 Ab binding to the neutrophil surface triggers a transient activation signal that requires extracellular calcium and regulates the adhesive activity of CD11/CD18. Associated protein kinase activity may play a role in signal transduction by CD63 to regulate other cell functions.
Reverse signaling via CD30 ligand.
Authors: Wiley S R , Goodwin R G , Smith C A
Published: January 2023

CD30 ligand (CD30L), a member of the TNF family, is a type II membrane protein with a C-terminal extracellular domain that is homologous with the extracellular domains of other TNF family members. Also, like most TNF family members, the N-terminal cytoplasmic domain of CD30L is conserved across species, but not between family members, suggesting a possible biological function. Motivated by this observation, we investigated the potential for CD30L, when activated by cross-linking, to directly transduce a signal to the ligand-bearing cell. Cross-linking of CD30L by a mAb or by CD30-Fc fusion protein induced the production of IL-8 by freshly isolated neutrophils. Further, both cross-linking mechanisms produced a rapid oxidative burst. Indirect effects through CD30 were ruled out, since CD30L, but not CD30, is expressed on neutrophils. Expression of CD30L can be induced in peripheral blood T cells by cross-linking the CD3 component of the TCR. Peripheral blood T cells exposed to suboptimal concentrations of anti-CD3 increased metabolic activity, proliferated, and produced IL-6 in response to cross-linking of CD30L. These results indicate that cross-linked CD30L can transduce a signal to the ligand-bearing cell. This "reverse signaling" via CD30L taken together with previously published data concerning other ligands in the TNF family strongly suggest that, as a rule, TNF family members and their cognate receptors signal bidirectionally, blurring the distinction between ligand and receptor.
Regulation of surface and soluble TNF receptor expression on human monocytes and synovial fluid macrophages by IL-4 and IL-10.
Authors: Hart P H , Hunt E K , Bonder C S , Watson C J , Finlay-Jones J J
Published: January 2023

By regulating monocyte and macrophage production of IL-1, its receptor, and its receptor antagonist, IL-4 and IL-10 may exert significant anti-inflammatory activity. We determined whether a similar multicomponent process controlling TNF activity was regulated by IL-4 and IL-10 in nonadherent monocytes and synovial fluid macrophages. Previous studies differed in their conclusions. For both the p75 and p55 TNF receptors, mRNA levels, surface receptor expression, and soluble receptor levels were measured for blood monocytes incubated in vitro for 17, 40, or 64 h with IL-4 or IL-10. The predominant TNF receptor on monocytes, the p75 receptor, was down-regulated by IL-4 at the mRNA level. In turn, both surface and soluble receptor levels on LPS-stimulated cells were reduced and the inhibitory effects were maintained for at least 64 h. In contrast, IL-10 increased surface and soluble p75 TNF receptor levels on monocytes for approximately 40 h, which reflected an increase in receptor mRNA. These studies suggest that IL-4 and IL-10 do not directly regulate the cleavage of TNF receptors from monocytes and macrophages. Addition of an Ab to IL-10 suggested that the stimulatory effects of LPS on p75 TNF receptor expression were due, at least in part, to LPS stimulation of IL-10 production and that IL-4 acted, in part, by decreasing IL-10 production. IL-4 was down-regulatory and IL-10 stimulatory for TNF receptor expression by synovial fluid macrophages. By increasing surface receptor levels, IL-10 enhanced the activities of TNF on monocytes for IL-1beta production. By increasing soluble TNF receptor levels, IL-10 may limit only temporarily the activity of other TNF-responsive cells. This study questions the benefit of IL-10 to resolving TNF-associated inflammation.
IL-15 stimulates the expansion of AIDS virus-specific CTL.
Authors: Kanai T , Thomas E K , Yasutomi Y , Letvin N L
Published: January 2023

It has been assumed that the maturation of pre-CTL to virus-specific effector CTL is dependent upon IL-2-mediated T cell triggering through the IL-2R. In view of its similarity to IL-2 in its effects on immune cells, we sought to determine whether IL-15 can induce the expansion of AIDS virus-specific pre-CTL to mature CTL. PBL of SIV(mac)-infected rhesus monkeys or HIV-1-infected humans have previously been shown to expand to effector CTL when cultivated with a predicted CTL epitope peptide and rIL-2. We now demonstrate that rIL-15 facilitates this expansion of effector CTL. In fact, rIL-15-driven expansion of virus-specific CTL occurs in the presence of IL-2-neutralizing or anti-IL-2R Abs, indicating that this cellular maturation can occur in an IL-2-independent fashion. These studies suggest a mechanism by which CTL may be capable of expanding in vivo in the absence of IL-2 and functional CD4+ T lymphocytes.
The immune response to 52-kDa Ro and 60-kDa Ro is linked in experimental autoimmunity.
Authors: Keech C L , Gordon T P , McCluskey J
Published: January 2023

Clustering of autoantibody specificities is a consistent finding in patients with systemic autoimmune diseases. Patients with Sjögren's syndrome frequently have autoantibodies to La, 60-kDa Ro(SS-A) protein (Ro60), and 52-kDa Ro(SS-A) protein (Ro52). In the case of anti-Ro60 and anti-La, there is evidence that these specificities occur together because of the physical association of the Ro60 and La proteins that form a ribonucleoprotein particle (RNP). Thus, the autoantibody response may spread from a single epitope to involve new epitopes located within other components of the RNP. The physical association of Ro52 with the Ro/La RNP has remained controversial, implying that Abs to Ro52 are not a consequence of intermolecular spreading and may be triggered independently of the anti-Ro60 response. To examine this relationship of the immune response to Ro52 and Ro60, mice were immunized with recombinant Ro52, Ro60, or La, and examined for autoantibody production. Immunization with Ro52 resulted in rapid, high titer Ab production to Ro52, followed 7 to 14 days later by lower titer autoantibody production to Ro60. Immunization with Ro60 led to anti-Ro60, which was also followed 7 to 14 days later by a lower titer anti-Ro52 response. Cross-reactivity of affinity-purified Abs from immune mouse sera was not observed. These observations suggest that the autoimmune responses to Ro60 and Ro52 are linked intrinsically, despite previous evidence suggesting they are not associated in vivo. The mechanism of linkage remains unclear, but the data are most consistent with some physical association of Ro52 and Ro60 allowing autoimmunization, presumably as a result of normal cell turnover or specific injury in vivo.
High density insulin receptor-positive T lymphocytes from nonobese diabetic mice transfer insulitis and diabetes.
Authors: McInerney M F , Flynn J C , Goldblatt P J , Najjar S M , Sherwin R S , Janeway C A
Published: January 2023

In the nonobese diabetic mouse, insulin-dependent diabetes is an autoimmune disease characterized by T cell-mediated invasion and destruction of pancreatic islet beta cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined, since it has been shown that the IR is a chemotactic receptor capable of directing cell movement in response to insulin. Using polyclonal antisera to the IR, phenotypic analysis of purified splenic T cells from diabetic mice showed that about 15% of T cells expressed high density IR (IRhigh). In addition, IRhigh T cells were already a dominant phenotype in the insulitis of young prediabetic mice. To determine the ability of IRhigh T cells to transfer diabetes, cells were sorted by flow cytometry before adoptive transfer into young (6- to 8-wk-old) nondiabetic irradiated nonobese mice. Transfer of as few as 3 x 10(6) purified IRhigh T cells alone resulted in rapid onset of insulitis and diabetes, and IRhigh-depleted T cells were essentially unable to passage either insulitis or diabetes. The adoptive transfer of disease was not due to the transfer of activated cells, since removal of IL-2R+ or transferrin R+ cells did not alter diabetes transfer. Therefore, IRhigh T cells are aggressively diabetogenic, suggesting that increased IR expression may provide a mechanism for delivering potentially autoreactive T cells to the islet, regardless of their activation state.
Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis.
Authors: Wakkach A , Guyon T , Bruand C , Tzartos S , Cohen-Kaminsky S , Berrih-Aknin S
Published: January 2023

The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG.
Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a T helper-1-like or a T helper-2-like response.
Authors: Sun B , Rizzo L V , Sun S H , Chan C C , Wiggert B , Wilder R L , Caspi R R
Published: January 2023

This study examines whether genetic susceptibility vs resistance to experimental autoimmune uveoretinitis (EAU) in mice is associated with dominant type 1 vs type 2 cytokine response profiles. Mice from six strains were immunized with the uveitogenic retinal Ag IRBP. EAU was evaluated by histopathology. As judged by disease scores, three of the strains were susceptible, one was minimally susceptible, and two were resistant. Ag-specific type 1 vs type 2 cytokine responses (protein and/or mRNA) in draining lymph node cells, and IgG2a vs IgG1 Ab isotypes to IRBP, were measured as indicators of Th1-like vs Th2-like responses, respectively. The three susceptible strains (B10.A, C57BL/10, and BALB/k) showed a dominant Th1-like response profile characterized by high IFN-gamma and IL-12p40 (but not IL-4) responses, and a predominance of IgG2a Abs. The minimally susceptible strain (A/J) had an IFN-gamma response detectable only at the mRNA level, but produced predominantly IgG2a Abs. One of the two resistant strains (BALB/c) showed a characteristic Th2-like response with dominant Ag-specific IL-4 and IL-10 responses but no IFN-gamma, and predominantly IgG1 Abs. However, the other resistant strain (AKR) did not show a Th2-dominated response pattern, in that it had low, or no, IL-4 and IL-10 responses, and made predominantly IgG2a Abs to IRBP. These results suggest that whereas a Th1 response is required for susceptibility, resistance is not dependent on a Th2 response pattern. We suggest that regulatory influences other than skewing the response toward the Th2 pathway may be equally effective at conferring genetic resistance to EAU.
B cell genotype determines the fine specificity of autoantibody in lpr mice.
Authors: Kakkanaiah V N , Sobel E S , MacDonald G C , Cheek R L , Cohen P L , Eisenberg R A
Published: January 2023

Anti-Sm Abs are specific markers of human systemic lupus erythematosus (SLE) and of murine models of this disease. In humans, anti-Sm Abs are mostly IgG1, and in MRL/lpr mice, IgG2a; both are T-dependent isotypes. Other lpr strains, such as B6/lpr, do not produce anti-Sm Ab spontaneously. The present study was aimed at identifying the cellular expression of background genes responsible for generation of the anti-Sm Ab response in MRL/lpr mice. We used double chimeric mice made by transferring MRL/lpr and B6/lpr bone marrows into irradiated allotype heterozygous F1 mice. Five mo after reconstitution, FACS analysis of lymph node (LN) and spleen cells revealed that both MRL/lpr and B6/lpr cells coexisted in roughly equal numbers. Ab produced by each donor could be distinguished by allotype-specific assays. IgG2a anti-Sm was made only by MRL-derived B cells despite the presence of T cells that might potentially provide help to the B6/lpr B cells. The frequency of anti-Sm Ab-producing individuals was similar to that of unmanipulated MRL/lpr mice (about 25%). IgG2a anti-chromatin and total IgG2a was mostly dominated by the MRL-derived B cells. B6-derived B cells produced more rheumatoid factor (RF) against their own IgG2b(b), while RF against IgG2a was dominated by MRL-derived B cells. This suggests that the control of the production of particular autoantibody specificities, such as anti-Sm, is determined by the expression of MRL or B6 background genes in B cells.
Reconstitution of the extrathymic intestinal T cell compartment in the absence of irradiation.
Authors: LefrançoisL  , Olson S
Published: January 2023

Extrathymic development of intestinal intraepithelial lymphocytes was studied using a reconstitution model that does not require irradiation. WBB6F1/J-Kit(W)/Kit(W-v) mice were reconstituted with normal fetal liver cells. In this system, reduced c-Kit activity in host hemopoietic progenitors imparts normal precursors with a growth advantage and, thus, chimerism can be established without irradiation. In control mice, TCR gammadelta and TCR alphabeta intraepithelial lymphocytes (IEL) developed efficiently from fetal liver cells, with a predominance of TCR alphabeta over TCR gammadelta IEL. In contrast, development in reconstituted thymectomized mice was heavily skewed toward TCR gammadelta IEL generation. In thymectomized mice, development of CD4+ 8- and CD4+ 8+ TCR alphabeta IEL did not occur, while TCR alphabeta CD8 alphabeta development was nearly absent. The results indicated that without irradiation the majority of TCR alphabeta IEL were thymus dependent, whereas TCR gammadelta IEL developed extrathymically. Thus, the discrepancies observed between different models of athymic development may be explained by the induction of T cell development as a result of irradiation.
Characterization of transport of newly assembled, T cell-stimulatory MHC class II-peptide complexes from MHC class II compartments to the cell surface.
Authors: Pond L , Watts C
Published: January 2023

In human B cells MHC class II molecules acquire antigenic peptides in lysosome-related compartments called the MHC class II compartments (MIIC). How assembled complexes, capable of activating T cells, then reach the cell surface has not been fully resolved. We have used selective ablation of early and recycling endosomes to determine whether newly peptide-loaded class II molecules require functional recycling endosomes to exit to the cell surface. Cellular compartments accessed by transferrin-horseradish peroxidase conjugates were functionally inactivated by cross-linking with diaminobenzidine and hydrogen peroxide. Cells with ablated endosomal compartments were unable to recycle transferrin to the cell surface and could not deliver exogenous Ag for processing and presentation to T cells. In contrast, cells that had taken up Ag and assembled intracellular class II-peptide complexes before endosome ablation were still able to deliver class II-peptide complexes to the cell surface and stimulate T cell proliferation. This delivery was abolished in the presence of brefeldin A. These data show that the parts of the endocytic apparatus necessary for Ag delivery to the MIIC are not required for functional class II-peptide complexes to reach the cell surface. Moreover, the brefeldin A sensitivity of this final step in the class II molecule biosynthetic pathway suggests a vesicular intermediate for transport between the MIIC and the plasma membrane.
The role of protein kinase C in CD8+ T lymphocyte effector responses.
Authors: Nesic D , Jhaver K G , Vukmanović S
Published: January 2023

Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumors but not syngeneic wild-type tumors. The lack of syngeneic tumor rejection in vivo is correlated with a partial response of beta2m-/- CD8+ cell lines to syngeneic tumor cells in vitro. This partial response is characterized by perforin/granzyme-mediated cytolytic activity in the absence of cytokine secretion or proliferation. Allogeneic tumors induce cytolysis, cytokine secretion, and proliferation. Cytokine secretion may therefore be an important effector mechanism for tumor rejection by CD8+ T cells. To determine the missing signaling events needed for cytokine secretion as well as the events inducing the isolated cytotoxic response, we attempted to restore cytokine secretion of beta2m-/- CD8+ cells to syngeneic MHC class I. Phorbol ester and syngeneic tumor cells acted synergistically to induce full responsiveness of beta2m-/- CD8+ cells. However, this synergistic induction of cytokine secretion used a different pathway than that induced by alloantigen. Protein kinase C (PKC) inhibitor prevented the syngeneic class I plus PMA-induced cytokine secretion, but not allo-class I-induced cytokine secretion. In contrast to the PKC independent alloantigen-induced cytokine secretion, cytolysis of both allogeneic and syngeneic targets was PKC dependent. The differential dependence of effector functions on PKC activation was also found in beta2m+/+ CD8+ T cells. Thus, two distinct signaling pathways (PKC dependent and PKC independent) may ultimately converge to induce cytokine secretion in CD8+ cells. The TCR engagement-initiated pathway is PKC independent, whereas the phorbol ester-activated pathway is PKC dependent.
Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells.
Authors: Pape K A , Khoruts A , Mondino A , Jenkins M K
Published: January 2023

Despite the wealth of information on the signals required for T cell activation in vitro, the signals required for the generation of functional Th cells in vivo are poorly understood. We addressed this by directly tracking the behavior of adoptively transferred CD4+ TCR transgenic T cells following Ag administration in vivo. Injection of soluble Ag induced a transient accumulation of Ag-specific T cells in lymphoid tissue. If bacterial LPS was present during this period, enhanced numbers of Ag-specific T cells accumulated, migrated into B cell-rich follicles, and provided help for Ab production. The ability of LPS to enhance the accumulation and follicular migration of Ag-activated T cells was mimicked by the proinflammatory cytokines, TNF-alpha and IL-1, and the capacity of LPS to promote the generation of IFN-gamma-secreting T cells, which provide help for IgG2a production, was mimicked by IL-12. Thus, the in vivo generation of functional Th cells can arise from Ag-dependent clonal expansion in the context of inflammatory cytokines.
The 25-kDa soluble CD23 activates type III constitutive nitric oxide-synthase activity via CD11b and CD11c expressed by human monocytes.
Authors: Aubry J P , Dugas N , Lecoanet-Henchoz S , Ouaaz F , Zhao H , Delfraissy J F , Graber P , Kolb J P , Dugas B , Bonnefoy J Y
Published: January 2023

CD23, a low-affinity receptor for IgE, was recently shown to bind to CD11b and CD11c molecules on human monocytes. The 25-kDa soluble fragment of CD23 (sCD23), was tested for its capacity to elicit various signaling pathways in human monocytes. sCD23 was found to trigger an early increase in cGMP accumulation, through the generation of nitric oxide. This was a result of activating the L-arginine pathway, since the sCD23-mediated effect was inhibited in the presence of substituted nonmetabolizable L-arginine analogues. In addition, the increase in cGMP was suppressed by calcium chelators and inhibitors of the calcium/calmodulin complex, suggesting the involvement of a constitutive, calcium-dependent nitric oxide synthase (NOS). Indeed, the presence of an endothelial constitutive type III NOS mRNA was detected in nonactivated human monocytes, and the corresponding protein has been detected by flow cytometry. Moreover, sCD23 was shown to induce a calcium influx in monocytes, in accordance with an activation of a constitutive NOS through a transient increase in [Ca2+]i. As expected, these events were mimicked by mAbs against CD11b and CD11c, the macrophage receptors for CD23. In addition to these early events, sCD23 and the agonistic anti-CD11b and CD11c mAbs, which all trigger the release of proinflammatory mediators such as TNF-alpha, were shown to act through an NO-dependent process.
Activation of human dendritic cells following infection with Mycobacterium tuberculosis.
Authors: Henderson R A , Watkins S C , Flynn J L
Published: January 2023

Dendritic cells (DC) play an essential role in the initiation of primary T cell responses to foreign Ag. It is likely that these potent APC are critical in the initiation of immune responses to pathogens, such as bacteria or parasites. However, little is known about the interaction of these important APC with pathogens. To address this issue, the interaction of the bacterium Mycobacterium tuberculosis with human DC was studied. DC generated from human peripheral blood by short term culture in medium containing recombinant human cytokines granulocyte-macrophage-CSF and IL-4 were capable of phagocytosing M. tuberculosis. Infection of DC with live M. tuberculosis bacilli resulted in increased APC surface expression of the costimulatory molecules CD54, CD40, and B7.1, as well as MHC class I molecules. In addition, infected DC secreted elevated levels of inflammatory cytokines, including TNF-alpha, IL-1, and IL-12. M. tuberculosis-infected human monocytes also secreted inflammatory cytokines, but exhibited no enhancement of costimulatory or MHC class I molecule expression. These data indicate that infection with M. tuberculosis results in the direct activation and maturation of these DC. In vivo, such activation may facilitate migration to the lymph nodes, and enhance presentation of Ag to T cells, thereby facilitating the induction of the immune response against this pathogen.
Migration and activation of antigen-specific CD8+ T cells upon in vivo stimulation with allogeneic tumor.
Authors: Kedl R M , Mescher M F
Published: January 2023

The response of CD8+ T cells to allogeneic tumor was studied by adoptive transfer of cells from TCR transgenic 2C mice specific for Ld alloantigen. Transferred cells were monitored during the course of a response to i.p. challenge with live P815 (H-2d) using the 1B2 mAb specific for the 2C TCR. Tumor was present in the draining LN and spleen within 3 to 4 days of challenge. The first changes in 1B2+ cells occurred in the spleen on day 4; VLA-4 expression increased in an Ag-specific manner and L-selectin expression decreased in an Ag-nonspecific manner. The number of 1B2+ cells in the spleen declined over days 4 to 6. The first detectable increase in CD25 expression and blast transformation was in the peritoneal cavity beginning days 5 and 6. Clonal expansion was largely limited to this site and was maximal on day 8. As expansion occurred in the peritoneal cavity; the number of 1B2+ cells in the draining LN and spleen also increased. These cells had an activated phenotype (CD44(high), VLA-4(high)) but most did not express CD25 and were not blasts. These results suggest that initial Ag recognition in the spleen results in altered expression of adhesion receptors so that cells gain access to the peritoneal cavity where they undergo clonal expansion and differentiation. Following the response, 1B2+ cells decline in number but a memory population (CD44(high), L-selectin(high and low)) persists for long times in the spleen and LN.
A new 12-kilodalton dimer associated with pre-TCR complex and clonotype-independent CD3 complex on immature thymocytes.
Authors: Takase K , Wakizaka K , von Boehmer H , Wada I , Moriya H , Saito T
Published: January 2023

We have characterized the pre-TCR/CD3 complex and the clonotype-independent CD3 (CIC) complex expressed on the cell surface of the immature cell line KKF which we previously identified as the cell line expressing only TCR beta-chain associated with the CD3 complex on the cell surface. We now show that KKF expresses the pT alpha-beta heterodimer-CD3 complex and also the CIC complex on the cell surface by surface biotinylation. In addition, not only the CIC complex but also the pT alpha-beta complex was found to be associated with the molecular chaperon calnexin. Whereas the CD3 zeta-chain was very weakly associated with the pre-TCR/CD3 complex, a 12-kDa molecule (designated pTAC12 as pre-TCR-associated chain) was more stably associated as a dimer with the pre-TCR complex as well as the CIC complex on KKF cells. The significance of pTAC12 dimer in the CD3 complex of immature thymocytes in vivo was demonstrated by coprecipitation of pTAC12 with the CD3 complex in thymocytes from SCID mice. These results show that pTAC12 is a component of the pT alpha-beta complex as well as the CIC complex, and they suggest that pTAC12 may play a role in signaling through and/or in the expression of the pre-TCR/CD3 complex.
CD50 monoclonal antibodies inhibit neutrophil activation.
Authors: Skubitz K M , Campbell K D , Skubitz A P
Published: January 2023

The constitutive high expression of CD50 (ICAM-3) on resting leukocytes, coupled with the observation that CD50 is the primary LFA-1 ligand on resting T cells, suggests that CD50 may be an important LFA-1 ligand in the initiation of the immune/inflammatory response. CD50 mAbs have been reported to increase homotypic adhesion of lymphocytes, and lymphocyte adhesion to HUVEC and extracellular matrix proteins. In this study, the effects of CD50 mAbs on neutrophil activation were examined. CD50 mAbs were found to inhibit neutrophil adhesion induced by FMLP and 12-O-tetradecanoyl-phorbol-13-acetate to resting and TNF-activated HUVEC. CD50 mAbs also inhibited neutrophil adhesion stimulated by CD66a, CD66b, CD66c, and CD66d mAbs to HUVEC. CD50 mAbs inhibited the up-regulation of CD11b/CD18 to the neutrophil surface, and the down-regulation of surface CD62L expression. The potential contribution of src family kinases to the previously described tyrosine kinase activity associated with CD50 in neutrophils was also examined. hck and lyn were found to account for much of the tyrosine kinase activity associated with CD50 in neutrophils. The data indicate that CD50 in neutrophils functions not only as a potential ligand for LFA-1, but also regulates the surface expression and activity of CD11b/CD18 and CD62L. In contrast to the effects in lymphocytes, CD50 appears to function as a negative regulator of neutrophil activation.
TNF-alpha-mediated expression of the receptor for anaphylatoxin C5a on neurons in experimental Listeria meningoencephalitis.
Authors: Stahel P F , Frei K , Eugster H P , Fontana A , Hummel K M , Wetsel R A , Ames R S , Barnum S R
Published: January 2023

The anaphylatoxin C5a has been implicated in the pathogenesis of bacterial meningitis as a potent mediator of inflammation in the subarachnoid space. We investigated the expression of the receptor for C5a (C5aR) in brains of mice with experimental Listeria monocytogenes (LM) meningoencephalitis. In the course of the disease, infiltrating cells in the meninges and the ventricles were found to express C5aR mRNA and protein. In the brain parenchyma, very low constitutive C5aR expression was seen on pyramidal neurons and Purkinje cells. However, in LM-infected mice, a dramatic increase in C5aR expression occurred on neurons starting 6 h after infection and was maximal between 24 and 36 h. TNF-alpha was identified as an essential mediator of neuronal C5aR expression, since mice lacking the genes for TNF and lymphotoxin-alpha (TNF/lymphotoxin-alpha -/- mice) showed significantly attenuated C5aR expression after LM infection. Furthermore, i.p. injection of recombinant TNF-alpha induced enhanced C5aR expression in the brains of TNF/lymphotoxin-alpha -/- mice and in normal animals even in the absence of a bacterial infection. We also assessed the levels of anaphylatoxin C5a in the cerebrospinal fluid of patients with infectious meningitis. C5a was detected in all patients with bacterial meningitis (n = 9), in 6 of 18 patients with aseptic meningitis, and in 1 of 66 control patients. The finding of TNF-alpha-mediated C5aR expression on neurons in experimental Listeria meningitis and the detection of the ligand, C5a, in the cerebrospinal fluid of human patients with infectious meningitis present new directions in the investigation of the pathophysiologic sequelae leading to secondary brain damage.
Chemokine expression in experimental tubulointerstitial nephritis.
Authors: Tang W W , Qi M , Warren J S , Van G Y
Published: January 2023

Chemokines may be important in the pathogenesis of leukocyte infiltration in tubulointerstitial nephritis associated with glomerular disease. We studied the renal cortical expression of the C-C (macrophage inflammatory protein-1alpha (MIP-1alpha)), monocyte chemotactic protein-1 (MCP-1), and RANTES) and C-X-C (interferon-inducible protein-10 (IP-10), MIP-2, and cytokine-induced neutrophil chemoattractant (CINC)) chemokines 4, 6, 8, 10, 14, and 21 days after the induction of puromycin aminonucleoside (PAN) nephrosis. There was a 7- to 10-fold increase in the steady state mRNA expression of IP-10 and MCP-1 in the renal cortex of rats 6 to 8 days after the administration of PAN that declines thereafter reaching control values by day 21. The site of IP-10 and MCP-1 mRNA production was localized to intrinsic tubulointerstitial cells and not to infiltrating monocytes or macrophages. By comparison, there was a low basal expression of RANTES mRNA in the renal cortex of nephrotic rats that did not differ from those of control rats. In contrast, CINC, MIP-2, and MIP-1alpha mRNAs were not detected. Translation of MCP-1 mRNA into protein was confirmed with an ELISA. These changes in chemokine gene expression were associated with a tubulointerstitial T lymphocyte and macrophage infiltration beginning on day 6 that peaked on day 10. Administration of a neutralizing Ab to rat MCP-1 (n = 5) beginning on day 4 resulted in a 45% decline in tubulointerstitial macrophage infiltration from 8.4 +/- 1.3% to 4.6 +/- 0.4% (p < 0.001) on day 6. These data provide evidence that MCP-1, and possibly IP-10, are important in the pathogenesis of monocyte/macrophage infiltration in the tubulointerstitial nephritis associated with PAN nephrosis.
Stimulation of Fc gamma receptors in rat peritoneal macrophages induces the expression of nitric oxide synthase and chemokines by mechanisms showing different sensitivities to antioxidants and nitric oxide donors.
Authors: Bayón Y , Alonso A , Sánchez Crespo M
Published: January 2023

The induction of nitric oxide (NO) production and the expression of cytokine-induced neutrophil chemoattractant (CINC-1) were studied in rat peritoneal adherent cells stimulated with insoluble immune complexes containing rabbit IgG Ab and OVA as the cognate Ag (IC). Incubation with IC at concentrations as low as 10 microg/ml induced NO production and the expression of inducible NO synthase (iNOS) protein. This was accompanied by the expression of CINC-1 mRNA and the activation of nuclear factor-kappaB (NF-kappaB). However, the expression of iNOS and CINC-1 mRNA induced by IC showed a different temporal pattern and a different sensitivity to both the antioxidant agent pyrrolidine dithiocarbamate (PDTC) and modulation by NO itself. Whereas iNOS mRNA and protein expression were blunted by PDTC and NO-generating compounds, CINC-1 mRNA expression was either enhanced or not affected by PDTC and NO donors. The time course of NF-kappaB activation was parallel to that of iNOS induction and was influenced in the same sense as iNOS induction by antioxidants, NO donors, the protease inhibitor N-tosyl phenylalanine chloromethyl ketone, and inhibitors of protein tyrosine phosphorylation reactions. These data indicate the existence in rat macrophages of a signaling mechanism triggered by Fc gammaR occupancy that leads to nuclear signaling, is initiated by protein tyrosine phosphorylation reactions, and shows specific sensitivities to antioxidants and NO. Whereas trans-activation of the iNOS gene can be fully explained by the stimulation of NF-kappaB, induction of CINC-1 mRNA expression seems influenced by additional regulatory elements.
Binding of human peripheral blood polymorphonuclear leukocytes to E-selectin (CD62E) does not promote their activation.
Authors: Repo H , Rochon Y P , Schwartz B R , Sharar S R , Winn R K , Harlan J M
Published: January 2023

E-selectin (CD62E) is a cytokine-inducible endothelial cell adhesion molecule that tethers polymorphonuclear leukocytes (PMNs) and supports PMN rolling under conditions of flow. We examined whether interaction of PMNs with E-selectin also leads to activation of CD11b/CD18 (Mac-1, alphaMbeta2), an event that can promote firm adhesion. PMNs were added to monolayers of IL-1beta-activated HUVECs and Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA. PMN activation was assessed by 1) increased CD11b/CD18 surface expression, 2) appearance of activation epitope on CD11b/CD18 (CD11b*) detected by mAb CBRM1/5, and 3) decreased L-selectin (CD62L) expression, as determined by flow cytometry. Both adherent and nonadherent supernatant PMNs became activated on IL-1beta-pretreated HUVECs. This activation was not affected by CD62E-blocking mAb P6E2. The activation state of PMNs adhered to CHO cells transfected with E-selectin cDNA was not increased over background and was similar to that of PMNs exposed to parent CHO cells. The findings were confirmed using confocal microscopy, which allowed staining of the cells for CD11b* in situ. In concert, the results suggest that PMN binding to E-selectin does not elicit inter-receptor signaling that could result in strengthening of PMN adhesion to endothelium.
Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes.
Authors: Steeber D A , Engel P , Miller A S , Sheetz M P , Tedder T F
Published: January 2023

L-selectin is a cell surface adhesion receptor that mediates leukocyte rolling along vascular endothelium at sites of inflammation and lymphocyte attachment to endothelial cells within peripheral lymphoid tissues. Since ligation of L-selectin through its ligand recognition region may mimic physiologic ligand binding, a new panel of mAbs that engaged a conserved ligand-binding region within the lectin domains of human, mouse, and rat L-selectin were generated using L-selectin-deficient mice. Indeed, appropriate ligation of L-selectin generated transmembrane signals that resulted in immediate intercellular adhesion following cell-cell contact of lymphocytes, neutrophils, and L-selectin cDNA-transfected cells. Ab binding to only some epitopes within the lectin domain of L-selectin induced adhesion, while mAb binding to numerous other epitopes or other domains of L-selectin had no effect. PPME (yeast polyphosphomonoester core polysaccharide), a complex carbohydrate that mimics the natural L-selectin ligand, also induced potent intercellular adhesion. The induction of intercellular adhesion required cellular energy, an intact cytoskeleton, and cytoplasmic kinase activity as well as the membrane proximal and cytoplasmic domains of L-selectin. Therefore, ligation of L-selectin through specific ligand-binding epitopes identified by the mAbs generated in this study can generate transmembrane signals. Signaling through L-selectin may enhance leukocyte-endothelial cell interactions by serving as an activation/priming step during rolling, thereby promoting subsequent firm cell-cell adhesion in the presence of inflammatory mediators.
Increased susceptibility to endotoxin shock in complement C3- and C4-deficient mice is corrected by C1 inhibitor replacement.
Authors: Fischer M B , Prodeus A P , Nicholson-Weller A , Ma M , Murrow J , Reid R R , Warren H B , Lage A L , Moore F D , Rosen F S , Carroll M C
Published: January 2023

Endotoxin shock is a life-threatening syndrome associated with a Gram-negative infection and mediated by a systemic inflammatory response. As a major effector of inflammation, the complement system has been implicated in both the pathogenesis and the protection from endotoxin shock. To clarify the role of complement in endotoxin shock, we have used mice totally deficient in either complement component C3 or C4. We found that both the C3- and C4-deficient mice were significantly more sensitive to endotoxin than wild-type controls. The endotoxin-challenged complement-deficient mice failed to clear endotoxin efficiently from the circulation and this led to excess consumption of C1 inhibitor protein (C1 INH), a major regulator of both complement and the contact system of blood coagulation. Replacement of C1 INH rescued the endotoxin-challenged complement-deficient mice from shock and death. These findings suggest a novel therapy for treatment of endotoxemia with C1 INH protein.