bluuebunny/crossref_metadata_2025 huggingface dataset:
Human mast cells activate fibroblasts: tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis.
Authors: Gruber B L , Kew R R , Jelaska A , Marchese M J , Garlick J , Ren S , Schwartz L B , Korn J H
Published: December 2022

The effect of human mast cells on fibroblast activity was studied using an organotypic skin-equivalent culture system. Human mast cell-1 (HMC-1) cells were embedded in a collagen gel with neonatal dermal fibroblasts at a ratio of 1:4; keratinocytes then were allowed to stratify above this composite culture. Analysis of type a1(I) procollagen mRNA synthesis by in situ hybridization revealed a substantial increase in mRNA levels in the presence of mast cells and especially following degranulation, induced by calcium ionophore A23187. Tryptase, a major product of human mast cells, could substitute for mast cells in this culture system, up-regulating procollagen mRNA synthesis. Tryptase pretreated with the specific protease inhibitor bis(5-amidino-2-benzimidazo-lyl)methane (BABIM) markedly attenuated the collagen mRNA up-regulation. Further studies revealed HMC-1 cell sonicates stimulated fibroblast chemotaxis and procollagen mRNA synthesis. Inhibition of HMC-1 sonicates with either BABIM or a neutralizing mAb against tryptase resulted in significant reduction of fibroblast chemotaxis and procollagen mRNA, implying that tryptase accounted for the majority of HMC-1 sonicate activity. Tryptase directly stimulated fibroblast chemotaxis with optimal concentrations between 10 pM and 1 nM. The maximal response of optimal concentrations of tryptase was comparable with the known fibrogenic factor, TGF-beta. Inhibition of tryptase with BABIM resulted in approximately 50% reduction in chemotactic activity. Additional studies revealed that tryptase (0.3-3 nM) stimulated procollagen mRNA synthesis in confluent monolayers of dermal fibroblasts.
Regulation of brain-derived T cells during acute central nervous system inflammation.
Authors: Irani D N , Lin K I , Griffin D E
Published: December 2022

The unique immunologic environment of the central nervous system (CNS) regulates most local inflammatory responses. In some circumstances, however, immune-mediated injury to the brain can occur. To understand how lymphocytes are regulated within the CNS during an inflammatory response that does not produce immunopathology, we have studied T cells isolated from the brains of mice with Sindbis virus (SV) encephalitis. Even though they express activation markers, these T cells are arrested in the cell cycle and do not proliferate in vitro. Altered phosphorylation of the retinoblastoma gene product, a critical cell cycle regulator, appears to mediate this effect. Furthermore, while brain-derived T cells generate IFN-gamma, IL-4, and IL-10, these T cells are deficient in IL-2 production compared with peripheral T cells. This pattern of cytokine production occurs in cells that do not activate NF-kappaB normally. When T cells producing both IL-2 and IFN-gamma are adoptively transferred into SV-infected mice, some of these cells traffic into the brain. Those that enter the brain selectively down-regulate IL-2 production over time. Since normal brain lipids can inhibit IL-2 production and T cell proliferation in vitro, these substances may mediate these same effects in vivo. Collectively, these data show that the local environment of the CNS during SV encephalitis exerts a complex regulatory effect on T cells that are recruited into the brain. We speculate that this effect serves to prevent excessive local T cell reactivity. Whether and how this regulation might fail in the setting of autoimmune neurologic disease remains to be explored.
An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with candidiasis.
Authors: Romani L , Mencacci A , Cenci E , Del Sero G , Bistoni F , Puccetti P
Published: December 2022

Granulocytes may serve immunoregulatory and effector roles in different limbs of the immune response to infection. Using live vaccine strain or virulent challenge in mucosal or systemic infection of mice with Candida albicans, we examined the effect of mAb-mediated depletion of neutrophils on the course of primary and secondary challenge and on development of CD4+ cell-dependent immunity. We obtained evidence of deleterious effects of neutrophil depletion occurring at the time of infection under all conditions of testing, both in naive and in previously immunized mice. In contrast, neutrophil depletion appeared to benefit the hosts late in the course of an overwhelming systemic infection. In an attempt to correlate neutrophil function with the nature of the T cell response, we tested the ability of neutrophils to produce cytokines associated with functionally distinct CD4+ Th cell responses to Candida. We found that neutrophils were endowed with the capacity to secrete IL-12 and IL-10 in vitro in response to the yeast. Neutrophil ablation early in the course of Th1-associated, self-limiting infection appeared to change the qualitative development of the T cell response, and rendered mice susceptible to infection. In addition to long recognized contributions to acute anti-candidal responses, these data suggest an important role for neutrophils both in initiation and in expression of Candida-specific immunity.
Combined diesel exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-specific IgE and skews cytokine production to a T helper cell 2-type pattern.
Authors: Diaz-Sanchez D , Tsien A , Fleming J , Saxon A
Published: December 2022

We have previously shown that in vivo nasal challenge with diesel exhaust particles (DEP) induces both quantitative and qualitative changes in local IgE production and stimulates generalized local cytokine production. We have now investigated the combined effects of intranasal challenge with DEP plus ragweed allergen on local humoral immune responses. We collected nasal lavages from ragweed sensitized subjects at different times after nasal challenge. As compared with challenge with ragweed alone, challenge with both DEP and ragweed induced markedly higher ragweed-specific IgE but not total IgE levels or IgE-secreting cell numbers. Total and specific IgG4 levels also were enhanced, while total IgG levels were not. Synergy was also observed between the DEP and ragweed in altering the profile of epsilon mRNAs generated by alternative splicing, mRNAs that code for different expressed IgE proteins. Intranasal challenge with ragweed alone induced inconsistent and low levels of mucosal cytokine mRNAs. In contrast, challenge with both ragweed plus DEP resulted in decreased expression for Th1-type cytokines (IFN-gamma and IL-2) but elevated expression of mRNA for other cytokines (IL-4, -5, IL-6, IL-10, IL-13). This synergy between DEP and natural allergen exposure is suggested as a key feature in increasing allergen-induced respiratory allergic disease.
Eosinophils are not required for the rejection of neovascularized fetal pig proislet xenografts in mice.
Authors: Simeonovic C J , Townsend M J , Wilson J D , McKenzie K U , Ramsay A J , Matthaei K I , Mann D A , Young I G
Published: December 2022

The rejection of neovascularized pig proislet (islet precursor) xenografts in mice is a CD4 T cell-dependent process involving invasion of the graft site mainly by host CD4 T cells and eosinophils. We previously identified CD4 T cell-dependent enhancement of intragraft IL-3, IL-4, and IL-5 mRNA expression during acute xeno-rejection in CBA/H recipient mice. In the present study we investigated the role of each cytokine and the involvement of eosinophils in the rejection of pig proislet xenografts using cytokine gene knockout mice (IL-4 -/- and IL-5 -/-) and the treatment of transplant recipients with anti-IL-3 mAb. In IL-4 -/- mice, IL-5 -/- recipient animals, and anti-IL-3 mAb-treated CBA/H mice, eosinophil accumulation at the transplant site was inhibited or ablated, but the kinetics of xenograft rejection was unaltered. Prolonged xenograft survival was only achieved in anti-CD4 mAb-treated mice and consistently correlated with the absence of intragraft IL-3, IL-4, and IL-5 mRNA enhancement. Together these findings indicate that neither IL-3, nor IL-4, nor IL-5 individually plays an obligatory role in the rejection process. The cytokine mRNA profile correlating with the lack of eosinophil recruitment was variable; the data suggest that IL-4 regulates eosinophil involvement in the xeno-rejection reaction indirectly via effects on IL-5 and IL-3 transcript expression. There is also suggestive evidence that IL-5 may influence IL-3 and IL-4 mRNA expression via feedback inhibition. Eosinophils, therefore, do not play an essential role in the rejection of neovascularized pig proislet xenografts in mice.
Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels.
Authors: Ridley S H , Sarsfield S J , Lee J C , Bigg H F , Cawston T E , Taylor D J , DeWitt D L , Saklatvala J
Published: December 2022

The role of p38 mitogen-activated protein kinase (MAPK) in responses of human fibroblasts and vascular endothelial cells to IL-1 was investigated by use of a pyridinyl imidazole compound (SB 203580), which specifically inhibits the enzyme. SB 203580 inhibited (50% inhibitory concentration approximately 0.5 microM) IL-1-induced phosphorylation of heat shock protein 27 (an indicator of p38 MAPK activity) in fibroblasts without affecting the other known IL-1-activated protein kinase pathways (p42/p44 MAPK, p54 MAPK/c-Jun N-terminal kinase and beta-casein kinase). SB 203580 significantly inhibited IL-1-stimulated IL-6, (30 to 50% at 1 microM) but not IL-8 production from human fibroblasts (gingival and dermal) and umbilical vein endothelial cells. IL-1 induction of steady state level of IL-6 mRNA was not significantly inhibited, which is consistent with p38 MAPK regulating IL-6 production at the translational level. SB 203580 strongly inhibited IL-1-stimulated PG production by fibroblasts and human umbilical vein endothelial cells. This was associated with the inhibition of the induction of PGH synthase-2 protein and mRNA. SB 203580 also inhibited the stimulation of collagenase-1 and stromelysin-1 production by IL-1 without affecting synthesis of the tissue inhibitor of metalloproteinases (TIMP)-1. SB 203580 prevented the increase in collagenase-1 and stromelysin-1 mRNA stimulated by IL-1. In a model of cartilage breakdown, short-term IL-1-stimulated proteoglycan resorption and inhibition of proteoglycan synthesis were unaffected by SB 203580, while longer term collagen breakdown was prevented. It is concluded that 1) p38 MAPK plays an important role in the regulation of some, but not all, responses to IL-1, and 2) it is involved in the regulation of mRNA levels of some IL-1-responsive genes.
Alterations in the level of CD45 surface expression affect the outcome of thymic selection.
Authors: Wallace V A , Penninger J M , Kishihara K , Timms E , Shahinian A , Pircher H , Kündig T M , Ohashi P S , Mak T W
Published: December 2022

CD45 is a receptor protein tyrosine phosphatase whose activity is required for thymocyte development and TCR-mediated signal transduction. Here we show that positive selection of TCR-alphabeta transgenic thymocytes is completely blocked in CD45 exon 6 -/- gene-deficient (CD45 -/-) mice that express the P14 TCR specific for the lymphocytic choriomeningitis virus. Thymocytes from mice heterozygous for the targeted disruption of the CD45 gene (CD45 +/-) displayed a reduction in both CD45 surface intensity and enzymatic CD45 protein tyrosine phosphatase activity. Surprisingly, positive thymocyte selection was enhanced in CD45 +/- mice as characterized by an up-regulation of the P14 TCR on thymocytes and increased numbers of transgenic T cells. Using a variant of lymphocytic choriomeningitis virus that is impaired in the induction of negative selection of P14 thymocytes, we also show that the reduction of CD45 surface expression in CD45 +/- mice rendered P14 transgene thymocytes susceptible to negative selection. These data demonstrate that changes in the expression level of the receptor protein tyrosine phosphatase CD45 can alter thymic selection.
Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein.
Authors: Yellen-Shaw A J , Wherry E J , Dubois G C , Eisenlohr L C
Published: December 2022

CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.
Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL.
Authors: Brossart P , Goldrath A W , Butz E A , Martin S , Bevan M J
Published: December 2022

Dendritic cells (DC) are potent inducers of CD8+ T cells and can stimulate protective antitumor immunity when pulsed with an antigenic peptide or protein. We used a replication-deficient adenovirus containing a Kb-restricted antigenic peptide of chicken OVA to study CTL induction in vitro and in vivo after adenovirus-mediated gene transfer into DC. The efficiency of adenovirus-infected DC in eliciting a specific CTL response was compared with immunizations with a recombinant vaccinia virus and DC pulsed with peptide or protein. An immortalized DC line derived from a C57BL/6 mouse and freshly isolated splenic DC from C57BL/6 mice were used in CTL induction. Virus-infected DC elicited the strongest Ag-specific CTL response in vitro and in vivo and induced protective antitumor immunity to a challenge with EG.7 tumors (EL-4 cell line expressing OVA). Direct immunization of mice with recombinant adenovirus resulted in the induction of high titers of neutralizing Abs, which precluded a boost of a CTL response after repeated inoculations. However, repeated injections of virus-infected DC induced only low titers of neutralizing Abs. Furthermore, the presence of neutralizing Abs specific for the virus did not affect the usefulness of infected DC as repeated applications of virus-infected DC boosted the CTL response even in mice previously infected with the recombinant vector. The use of DC infected with a recombinant virus has advantages over other forms of immunization and could provide an alternative approach for designing vaccination therapies.
Borrelia burgdorferi activates nuclear factor-kappa B and is a potent inducer of chemokine and adhesion molecule gene expression in endothelial cells and fibroblasts.
Authors: Ebnet K , Brown K D , Siebenlist U K , Simon M M , Shaw S
Published: December 2022

Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi, is a systemic infection with preponderance for the skin, joints, heart, and nervous system. Inflammatory lesions of target organs are characterized by the presence of spirochetes and inflammatory leukocytes. We have analyzed the potential of B. burgdorferi to induce gene expression of chemokines and adhesion molecules in human endothelial cells, keratinocytes, and fibroblasts. We find induction of the chemokines RANTES (regulated upon activation, normal T cells expressed and secreted), monocyte chemoattractant protein-1, IL-8, gro-alpha, IFN-inducible protein-10, and mig (monokine induced by gamma-IFN), and of the adhesion molecules E-selectin, ICAM-1, and VCAM-1 in endothelial cells and induction of the same chemokines and ICAM-1 in fibroblasts. This is mediated by the lipid moiety of the outer surface lipoprotein A. Induction of chemokine and adhesion molecule genes by B. burgdorferi occurs rapidly and does not require new protein synthesis. Induction is blocked by inhibitors of nuclear factor (NF)-kappa B. We also find that B. burgdorferi induces nuclear translocation of NF-kappa B and a transient increase in the expression of its inhibitor I kappa B-alpha. These findings indicate that B. burgdorferi is a potent inducer of molecules required for leukocyte recruitment to inflammatory foci, and the data suggest that this biologic activity is due to the ability of the spirochetes to activate the pleiotropic transcription factor NF-kappa B.
Interleukin-13 induces human monocyte/macrophage fusion and macrophage mannose receptor expression.
Authors: DeFife K M , Jenney C R , McNally A K , Colton E , Anderson J M
Published: December 2022

Inasmuch as we recently demonstrated that IL-4 is a strong inducer of monocyte/macrophage fusion and IL-13 has been observed to mimic many of the biologic effects of IL-4, the ability of IL-13 to promote human macrophage fusion in vitro was tested and compared with IL-4-mediated fusion. IL-13 induced the fusion of monocyte-derived macrophages as potently as IL-4 under identical culture conditions, and resulted in foreign body-type giant cell formation. At optimal concentrations of cytokine added, statistically equal numbers of macrophages participated in IL-13- and IL-4-induced fusion (66.1 +/- 4.6% and 63.9 +/- 4.4%, respectively). However, the effects of IL-13 and IL-4 were not additive or synergistic, and the maximum fusion obtained when both IL-4 and IL-13 were added was 63.8 +/- 3.6%. Only anti-human IL-13 Abs inhibited IL-13-induced foreign body giant cell formation; the fusion-inducing effects of IL-13 continued to be observed in the presence of neutralizing Abs to IL-4 and several other anti-cytokine Abs, including Abs against IFN-gamma, granulocyte-macrophage CSF, IL-3, and TNF-alpha. IL-13 also significantly enhanced the fluorescence intensity detected by anti-human macrophage mannose receptor Abs, indicating that IL-13, like IL-4, up-regulates expression of the receptor that may be an essential participant in macrophage fusion. The results of this study demonstrate that IL-13, like IL-4, is a potent human macrophage fusion factor, and suggest that although IL-13 acts independently of IL-4 to promote foreign body giant cell formation, it may trigger a common mechanism for macrophage fusion.
Requirement for C-X-C chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) in IgG immune complex-induced lung injury.
Authors: Shanley T P , Schmal H , Warner R L , Schmid E , Friedl H P , Ward P A
Published: December 2022

The C-X-C chemokines of the IL-8 family possess potent chemotactic activity for neutrophils, but their in vivo role in inflammatory responses is not well understood. In the IgG immune complex-induced model of acute lung inflammatory injury in the rat we have evaluated the roles of two rat chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC). Both mRNA and protein for MIP-2 and CINC appeared in a time-dependent manner after initiation of IgG immune complex deposition in lung. There exists a 69% homology between the amino acid sequences for these proteins, and we found cross-reactivity between polyclonal Abs raised to these chemokines. By purifying the blocking Abs using double affinity methods (with Ag-immobilized beads), this cross-reactivity was removed. Individually, anti-MIP-2 and anti-CINC Ab significantly reduced lung injury (as measured by 125I-labeled albumin leakage from the pulmonary vasculature) and reduced neutrophil accumulation in the lung (as determined by myeloperoxidase (MPO content) and neutrophil counts in bronchoalveolar lavage (BAL) fluids); however, no change in TNF-alpha levels in BAL fluids was found. Chemotactic activity in BAL fluids collected 2 h after injury from animals undergoing immune complex deposition could be shown to be chiefly due to the combined contributions of MIP-2 (39%), CINC (28%), and C5a (21%). When either MIP-2 or CINC was blocked in vivo, up-regulation of Mac-1 expression on neutrophils obtained from BAL fluids was significantly reduced. These data suggest that, in the model studied, both MIP-2 and CINC contribute significantly to the influx of neutrophils and their activation.
Autoreactive T cell clones from patients with systemic lupus erythematosus support polyclonal autoantibody production.
Authors: Takeno M , Nagafuchi H , Kaneko S , Wakisaka S , Oneda K , Takeba Y , Yamashita N , Suzuki N , Kaneoka H , Sakane T
Published: December 2022

This work examines the functional properties and TCRbeta gene utilization of 15 autoreactive T cell clones derived from five patients with systemic lupus erythematosus. All these clones proliferated and secreted cytokine when stimulated in vitro by autologous (but not allogenic) B cells. Individual T cell clones used diverse TCRbeta genes and did not show skewing toward the preferential usage of anionically charged receptors. Autoreactive T cell clones supported polyclonal B cell activation, as characterized by the production of anti-DNA, anti-Sjögren syndrome A, and anti-tetanus toxoid (anti-TT) Abs. This T cell help was mediated through the production of immunostimulatory cytokines, especially IL-6. Although stimulation of the autoreactive clones was blocked by anti-HLA class II Abs, the T cell clones did not proliferate, nor did they support polyclonal IgG production by HLA class II-matched normal B cells. Unlike the autoreactive clones, TT-specific clones derived from the same patients provided help selectively to B cells secreting anti-TT Abs. These findings suggest that autoreactive T cells from systemic lupus erythematosus patients are triggered to provide help following cognate interactions with self-peptides presented in the context of HLA class II molecules expressed on autologous B cells regardless of their specificities.
Thymus-independent generation of NK1+ T cells in vitro from fetal liver precursors.
Authors: Shimamura M , Ohteki T , Launois P , Garcia A M , MacDonald H R
Published: December 2022

NK1.1+TCR alpha beta+ (NK1+) T cells are an unusual subset of mouse TCR alpha beta+ cells found primarily in adult thymus and liver. In contrast to conventional TCR alpha beta+ cells, NK1+ T cells have a TCR repertoire that is highly skewed to V alpha14 and to Vbeta8, -7, and -2. The developmental origin and ligand specificity of NK1+ T cells are controversial. We show here that NK1+ T cells with a typically biased V alpha and V beta repertoire develop in cytokine-supplemented suspension cultures of fetal liver established from either normal or athymic mice. Furthermore, NK1+ T cell development in fetal liver cultures is abrogated in beta2m-deficient mice (which lack MHC class I and other related molecules) and can be partially inhibited by the presence of anti-CD1 mAbs. Moreover, mixing experiments indicate that recombination-deficient SCID fetal liver cells can reconstitute NK1+ T cell development in beta2m-deficient fetal liver cultures. Collectively, our data demonstrate that NK1+ T cells can develop extrathymically from fetal liver precursors and that a beta2m-associated ligand (putatively CD1) present on nonlymphoid cells is essential for their positive selection and/or expansion.
Complement evasion by Echinococcus granulosus: sequestration of host factor H in the hydatid cyst wall.
Authors: Díaz A , Ferreira A , Sim R B
Published: December 2022

Hydatid disease, which affects humans and various livestock species, is caused by the larval stages of the cestode parasite Echinococcus granulosus. The disease involves the growth of a fluid-filled cyst in the host's internal organs. The hydatid cyst wall is generally thought to preclude the access of host immune cells to the cyst, while allowing passage of macromolecules. Thus, evasion of humoral effector mechanisms, in particular the complement system, may be crucial to parasite survival. Evasion of complement might involve inhibition of the C3b deposition step, which can in principle be achieved through parasite-derived or host-derived and parasite-bound regulatory molecules. In this work, extracts from bovine hydatid cysts were analyzed for the presence of factor H-like function, namely the cofactor activity for the breakdown of amidated bovine C3 by bovine factor I. Both this activity and antigenically detectable bovine factor H were found in most extracts. The cofactor activity was retained by an anti-bovine factor H Ab column; as no factor H-cross-reactive parasite molecules were detected in the active extracts, the activity was indeed due to host factor H. The pattern of extraction of host factor H from the parasite tissue indicated that at least part of it was selectively bound to the cyst wall, and that a strong charge component was involved in the binding. Our results indicate that the selective concentration of host factor H from plasma/interstitial fluid may contribute to complement evasion by E. granulosus.
Constitutive shedding of both p55 and p75 murine TNF receptors in vivo.
Authors: Pinckard J K , Sheehan K C , Arthur C D , Schreiber R D
Published: December 2022

Recently, we reported the generation and characterization of hamster mAbs specific for the murine p55 and p75 TNF receptors. Upon characterizing these mAbs in vivo, we discovered that administration of TNF receptor-specific mAb to normal mice resulted in the linear accumulation of the appropriate class of soluble TNF receptor in the circulation. The mechanism underlying soluble receptor accumulation was found to be due to an abrogation of the clearance of constitutively shed soluble receptor by receptor-specific mAb. Levels of p55 or p75 accumulated in the presence of nonblocking, nonagonistic TNF receptor mAb were capable of inhibiting murine TNF-induced responses in vivo. These results document that both p55 and p75 are constitutively shed in substantial amounts in vivo and suggest that the process of constitutive TNF receptor shedding plays an important role in regulating TNF activity under physiologic conditions.
Neutrophil-activating protein ENA-78 and IL-8 exhibit different patterns of expression in lipopolysaccharide- and cytokine-stimulated human monocytes.
Authors: Schnyder-Candrian S , Walz A
Published: December 2022

The production of epithelial neutrophil-activating protein-78 (ENA-78) by normal human monocytes was studied in comparison with IL-8 upon stimulation with various stimuli. LPS at 100 ng/ml was a strong inducer of ENA-78, yielding a delayed up-regulation of steady state mRNA peaking at 24 h and causing a long-lasting ENA-78 protein secretion starting 20 h after induction. As shown by specific ELISA and immunoprecipitation, ENA-78 secretion by monocytes was not down-regulated for up to 72 h. Thus, ENA-78 production becomes effective when IL-8 synthesis is shut off, since IL-8 mRNA peaks at 4 to 12 h and approaches background levels at about 16 h. Induction of steady state ENA-78 mRNA by proinflammatory cytokines IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml) was weaker than that by LPS and yielded a biphasic kinetic with a first maximum at 8 to 12 h and a second at 20 to 28 h. Steady state IL-8 mRNA induced by LPS, IL-1beta, or TNF-alpha was superinduced or unchanged in the presence of cycloheximide (10 microg/ml). In contrast, ENA-78 mRNA was completely abrogated, suggesting the involvement of a newly synthesized protein intermediate necessary for ENA-78 up-regulation. Dexamethasone treatment reduced ENA-78 mRNA and protein levels by 60% of the LPS control level. This inhibition was identical when dexamethasone was added 8 h after LPS induction. These results demonstrate significant differences in the production of monocyte-derived IL-8 and ENA-78 and thus may suggest different roles for the two chemokines in acute or chronic inflammation.
Differential chemotactic activities of sensory neuropeptides for human peripheral blood mononuclear cells.
Authors: Schratzberger P , Reinisch N , Prodinger W M , Kähler C M , Sitte B A , Bellmann R , Fischer-Colbrie R , Winkler H , Wiedermann C J
Published: December 2022

We studied the chemotactic effects of calcitonin gene-related peptide, vasoactive intestinal peptide, substance P (SP), and secretoneurin on PBMC and PBL using micropore filter assays. All four peptides induced migration of PBMC, whereas only calcitonin gene-related peptide, vasoactive intestinal peptide, and SP were chemotactic for PBL. Secretoneurin, known to induce monocyte chemotaxis, was unable to affect lymphocyte migration. Effects of SP on PBL were characterized by checkerboard analyses and represented true chemotaxis. Both T and B cells responded chemotactically to SP, the functional activity of SP residing in its C-terminal amino acid sequence. Involvement of neurokinin (NK) receptors was supported by inhibition of SP-induced migration of PBL with an NK1 receptor antagonist and induction of migration with [Sar9, Met(O2)11]SP and [PyrGlu6, Pro9]SP(6-11), two specific agonists for NK1 receptors, but not with [beta-Ala8]NK A(4-10), an agonist for NK2 receptors. PBL chemotaxis to SP was abolished by inhibition of tyrosin kinase but not by that of protein kinase C. Preincubation of PBL with pertussis or cholera toxin inhibited SP chemotaxis, indicating that in PBL, NK receptors for chemotaxis probably are coupled with G protein and involve a tyrosin kinase signaling pathway. We conclude that, together with calcitonin gene-related peptide and vasoactive intestinal peptide, SP is a lymphocyte chemoattractant, whereas secretoneurin, which is coreleased from sensory nerve endings, is not.
Chemokines are expressed in a myeloid cell-dependent fashion and mediate distinct functions in immune complex glomerulonephritis in rat.
Authors: Wu X , Dolecki G J , Sherry B , Zagorski J , Lefkowith J B
Published: December 2022

Using anti-glomerular basement membrane nephritis in rats, we investigated the mechanisms underlying in situ chemokine expression and the in vivo function of these cytokines during the acute phase of this model. We observed that CXC chemokine expression was monophasic and paralleled neutrophil (PMN) influx, whereas CC chemokine expression was biphasic with peaks coinciding with the influx of PMNs and macrophages (Mphi). The initial peak of chemokine expression was attenuated by decomplementation, neutropenia, and leukopenia, while the latter peak was attenuated only by leukopenia and augmented in the accelerated form of this disease model, corresponding to an increase in Mphi influx. Differential expression of chemokines by PMNs and Mphi was not an intrinsic property of these cells, as these leukocytes expressed similar profiles of chemokines in vitro. Immunostaining for Mphi inflammatory protein-1alpha, a CC chemokine, in acute nephritis validated that expression during acute nephritis was accompanied by local protein production. Moreover, neutralizing Ab to Mphi inflammatory protein-1alpha attenuated the acute phase proteinuria, but not the accompanying influx of PMNs. Neutralizing Ab to cytokine-induced neutrophil chemoattractant (a CXC chemokine), in comparison, inhibited both PMN influx and proteinuria. A combination of both Abs was not significantly more effective than either alone. In sum, the influx of myeloid cells is necessary for local chemokine expression in anti-glomerular basement membrane nephritis, although the differential expression of CXC and CC chemokines must involve additional factors. CXC and CC chemokines also mediate distinct, but overlapping, pathophysiologic roles in the acute phase of this model.
Lipopolysaccharide-binding protein and soluble CD14 transfer lipopolysaccharide to phospholipid bilayers: preferential interaction with particular classes of lipid.
Authors: Wurfel M M , Wright S D
Published: December 2022

LPS-binding protein (LBP) catalyzes the movement of LPS (endotoxin) from micelles directly to high density lipoprotein (HDL) particles, and this activity results in neutralization of the biologic activities of LPS. LBP also catalyzes the transfer of LPS to HDL by a two-step mechanism in which LPS is transferred to soluble CD14 (sCD14), and then from LPS-sCD14 complexes to HDL. In this work, we show that the phospholipid component of HDL, phosphatidylcholine (PC), is both necessary and sufficient for LBP-catalyzed neutralization of LPS through either mechanism. Our observation that LBP and sCD14 can transport LPS into phospholipid bilayers suggests that LBP and membrane CD14 may transport LPS into the phospholipid bilayer of cells such as monocytes and neutrophils. Studies with a variety of purified phospholipids showed that: 1) PC, phosphatidylserine, phosphatidylinositol, and sphingomyelin can neutralize LPS, while phosphatidylethanolamine, ceramide, and lactosylceramide cannot. 2) PC containing saturated long chain acyl groups (distearoyl-PC) does not neutralize LPS, but PC containing unsaturated long chain acyl groups (dioleoyl-PC) rapidly neutralizes LPS. 3) Inclusion of sCD14 is absolutely necessary to observe LBP-dependent neutralization of LPS by sphingomyelin, globoside, and phosphatidylserine. 4) Inclusion of sCD14 enhances movement to longer chain PC vesicles, but slows movement to certain short chain vesicles. These findings indicate that LBP and sCD14 will rapidly transfer LPS to certain membranes based on the kinetics of the movement of LPS into these membranes. This discrimination may target LPS to certain classes of lipoprotein, certain cell types, or even certain lipid domains at the cell surface.
Endotoxin and IL-1 hyporesponsiveness in a patient with recurrent bacterial infections.
Authors: Kuhns D B , Long Priel D A , Gallin J I
Published: December 2022

We describe a 15-yr-old girl with recurrent bacterial infections who is refractory to the effects of LPS in vivo and in vitro and IL-1 in vitro. Intravenous challenge of the patient with Escherichia coli endotoxin caused a subnormal febrile response, little alteration in the number of circulating neutrophils, and subnormal elevations in the plasma levels of TNF-alpha, IL-6, IL-8, lactoferrin, and granulocyte CSF; however, normal levels of the anti-inflammatory mediators IL-1 receptor antagonist and soluble TNF receptor (60 kDa) were induced. Studies in vitro indicated the patient's monocytes expressed CD14, the LPS receptor, and bound LPS in a specific manner, but failed to produce TNF-alpha and granulocyte CSF after stimulation with LPS, and failed to respond to IL-1, heat-killed Staphylococcus aureus, and soluble glucan. Peripheral blood patient neutrophils exhibited normal expression of CD14, but failed to respond to treatment with LPS (100-1000 ng/ml for 30 min at 37 degrees C), a treatment that caused increased expression of the surface markers, C10, CD18, CD11b, CD67, and CD45, and decreased expression of L-selectin in normal neutrophils. Treatment of normal and patient neutrophils with FMLP (0.1 microM) resulted in equivalent altered expression of these surface markers. Patient neutrophils could not be primed by either LPS or IL-1beta for enhanced FMLP-induced O2- generation, but primed normally to TNF-alpha and platelet-activating factor. This patient's hyporesponsiveness to LPS and IL-1 is most likely due to a defect very early in the signal-transduction pathway.
HIV-induced T cell syncytia are self-perpetuating and the primary cause of T cell death in culture.
Authors: Sylwester A , Murphy S , Shutt D , Soll D R
Published: December 2022

Prior studies suggested that induced fusion in T cell cultures infected with syncytium-inducing HIV was not the primary cause of T cell death. However, these studies failed to assess the contribution of fusion in terms of syncytium volume, rather than syncytium frequency. This question has been reassessed by monitoring the frequency, volume, rate of growth, longevity, p24 production, viral budding, and self-propagating ability of syncytia in HIV-infected SUP-T1 cell cultures and individually isolated syncytia seeded in uninfected SUP-T1 cell cultures. The results demonstrate that in these cultures syncytium formation is the principal mechanism of T cell death, syncytia are the main source of virus production, and both virus production and syncytium longevity are independent of syncytium size, but dependent on syncytium age, suggesting that both are programmed events.
Different developmental arrest points in RAG-2 -/- and SCID thymocytes on two genetic backgrounds: developmental choices and cell death mechanisms before TCR gene rearrangement.
Authors: Diamond R A , Ward S B , Owada-Makabe K , Wang H , Rothenberg E V
Published: December 2022

To analyze the early development of T cell precursors in the absence of TCR gene rearrangement, recombinase-activating gene-deficient (RAG-2 -/-) thymocytes were compared with thymocytes from SCID mice on the C.B-17 (BALB) and B6 genetic backgrounds. RAG-2 -/- thymocytes accumulate as quiescent cells with a heat-stable Ag (HSA)-positive CD25+ CD44- c-kit(low) phenotype, resembling normal cells just before selection for functional TCR beta-chain expression. CD44 and c-kit progressively down-regulate in the HSA+ subset, providing a background-independent and TCR-independent developmental clock. On this basis, compared with RAG-2 -/- thymocytes, SCID thymocytes 1) arrest at more heterogeneous, and generally earlier, stages; 2) accumulate to lower overall cell numbers; and 3) maintain higher populations of cycling and activated G1 cells, showing both increased responsiveness and increased cell death. B6-SCID thymocytes appear to die particularly early. Low levels of Fas were observed on "advanced" HSA+ SCID thymocytes but not on any RAG-2 -/- thymocytes, suggesting a potential difference in activation state or mechanism of death. In both RAG-2 -/- and SCID thymocytes, there are also two discrete subsets of HSA(low) CD25- CD44+ c-kit+ cells: a Sca-1+ CD44++ CD122- NK1.1- putative progenitor subset and an NK-like Sca-1- CD44+(+) CD122+ NK1.1+ subset. The absolute cell numbers in these HSA(low) subsets and the extent of NK cell differentiation, measured by perforin expression, are nearly constant in all the mutant strains analyzed, in contrast to the HSA+ CD25+ population, which was expanded in the RAG-2 -/-. Thus, the SCID thymocytes appear to undergo a normal generation but a premature death as compared with the RAG-2 -/- thymocytes.
Lck is necessary and sufficient for Fas-ligand expression and apoptotic cell death in mature cycling T cells.
Authors: Gonzalez-Garcia A , R-Borlado L , Leonardo E , Merida I , Martinez-A C , Carrera A C
Published: December 2022

Apoptotic cell death is induced in mature cycling T cells upon ligation of the Ag-specific TCR. This process is essential for the maintenance of homeostasis in the immune system, as it is capable of down-regulating ongoing immune responses. The analysis of the mechanism underlying TCR-induced programmed cell death has focused the attention of many scientists recently. In this regard, several recent reports have implicated Fas/Fas-ligand molecules as the final mediators of this process. Several other gene products have been implicated in the control of apoptosis (as Bcl-2, p53, and c-Myc); however, no information was available in the early signaling molecules that trigger this phenomena. The results presented in this work indicate that pp56(lck) src family kinase is actually required for the TCR to trigger cell death in mature cycling T cells. In fact, while inhibition of pp56(lck) expression with antisense oligonucleotides blocked TCR-induced apoptosis, pharmacologic inhibition of phosphatidylinositol 3-kinase activity had no effect. Accordingly, ligation of the Ag receptor in a cell line defective for pp56(lck) expression was unable to induce apoptosis, although it induced cellular stimulation, as measured by the expression of CD69. In addition, we show in this work that expression of constitutively active pp56(lck) mutants, but not pp59(fyn) mutants, in the absence of any other TCR-derived signal, is sufficient to induce apoptosis not only in transformed, but also in normal cycling T cells. Finally, evidence is presented indicating that a mechanism through which pp56(lck) regulates TCR-induced apoptosis in mature cycling T cells is by controlling Fas-ligand expression.
CD8Tc1 and Tc2 cells secrete distinct cytokine patterns in vitro and in vivo but induce similar inflammatory reactions.
Authors: Li L , Sad S , Kägi D , Mosmann T R
Published: December 2022

Naive CD8 T cells, similar to CD4 T cells, can differentiate into at least two subsets of cytolytic effector cells with distinct cytokine patterns: T cytotoxic-1 (Tc1) cells secrete a Th1-like cytokine pattern, including IL-2 and IFN-gamma; and Tc2 cells produce Th2 cytokines, including IL-4, IL-5, and IL-10. As CD4 Th1 cells induce delayed-type hypersensitivity (DTH) more effectively than Th2 cells, we tested the potential ability of Tc1 and Tc2 cells to induce DTH. Allospecific Tc1 or Tc2 cells were injected into the footpads of naive mice expressing the target Ag. Tc1 and Tc2 cells induced comparable levels of Ag-specific footpad swelling with similar kinetics. They also induced similar levels of footpad edema and similar infiltration of macrophages and neutrophils. However, Tc2 cells induced slightly more eosinophil infiltration. Analysis of footpad extracts showed that Tc1 and Tc2 cells retained their distinct in vitro cytokine profiles in the injected footpads. These results suggest that both Tc1 and Tc2 cytokines can be associated with the DTH reaction induced by CD8 T cells. Perforin-deficient Tc1 or Tc2 cells also induced DTH, although at lower levels, suggesting that perforin-mediated cytotoxicity of CD8 T cells is not essential for CD8-induced DTH. Thus, despite their distinct cytokine profiles in vitro and in vivo, Tc1 and Tc2 cells induce similar DTH reactions.
Antigen-specific B cells preferentially induce CD4+ T cells to produce IL-4.
Authors: Macaulay A E , DeKruyff R H , Goodnow C C , Umetsu D T
Published: December 2022

The role of Ag presentation by B cells in regulating the development of T cells with restricted cytokine profiles remains controversial. In this report, we compared Ag presentation by naive polyclonal B cells, naive Ag-specific B cells (from Ig receptor transgenic mice), or splenic adherent cells (SAC) and examined the capacity of these cells to influence cytokine production by CD4+ T cells. Freshly isolated naive B cells stimulated vigorous T cell proliferation and very strong T cell cytokine responses, but only when cultured with Ag recognized by the B cell Ig receptor (cognate Ag) and not when cultured with a noncognate Ag. Under these conditions, B cells activated by Ig receptor-mediated endocytosis of Ag induced both naive and Ag-primed CD4+ T cells to produce high levels of IL-4 (300-4000 pg/ml). In contrast, SAC induced the production of very low levels of IL-4 (<100 pg/ml) but much higher maximal levels of IFN-gamma than did Ag-specific B cells. The induction of IL-4 synthesis by Ag-specific B cells was significantly reduced by blocking CD40-CD40 ligand (CD40L) interactions or by the addition of small quantities of rIL-12. These results suggest that B cells activated by their cognate Ag preferentially induce IL-4 synthesis as a result of the interaction of CD40L on T cells with CD40, whereas SAC preferentially induce IFN-gamma synthesis by T cells as a result of their greater production of IL-12 and their limited capacity to trigger CD40L on T cells.
Differential expression of the beta2-adrenergic receptor by Th1 and Th2 clones: implications for cytokine production and B cell help.
Authors: Sanders V M , Baker R A , Ramer-Quinn D S , Kasprowicz D J , Fuchs B A , Street N E
Published: December 2022

An important function of the sympathetic nervous system is to maintain homeostasis by modulating the level of cellular activity in many diverse organ systems. The sympathetic neurotransmitter norepinephrine modulates the level of T and B lymphocyte activity by binding to the beta2-adrenergic receptor (beta2AR). The present study was designed to elucidate the mechanism by which stimulation of the beta2AR affects both Th1/Th2 cell cytokine production and Th1/Th2 cell-dependent Ab production. Clones of murine Th1/Th2 cells were exposed to the beta2AR agonist terbutaline before activation by Ag-presenting B cells. Terbutaline exposure of Th1 cells before activation inhibited IFN-gamma production by Th1 cells and subsequent IgG2a production by B cells. IgG2a inhibition was prevented by addition of the betaAR antagonist nadolol or exogenous IFN-gamma. In contrast to Th1 cells, terbutaline did not affect either IL-4 production by Th2 cells or subsequent IgG1 production by B cells. Although baseline levels of intracellular cAMP were similar in both subsets, terbutaline induced an increase in cAMP levels in Th1 cells only. Radioligand binding studies showed that a detectable number of beta2AR binding sites were present on Th1 cells, but not on Th2 cells. Immunofluorescence analyses showed that Th1 cells expressed a higher level of the beta2AR cytoplasmic carboxyl terminus than did Th2 cells. These results show that expression of the beta2AR binding site by Th1 cells, but not by Th2 cells, establishes a physiologic mechanism for selective modulation of Th1 cell IFN-gamma production and IFN-gamma-dependent IgG2a production, provided that beta2AR stimulation occurs before cell activation by a B cell.
Bone marrow-derived dendritic cells can process bacteria for MHC-I and MHC-II presentation to T cells.
Authors: Svensson M , Stockinger B , Wick M J
Published: December 2022

Dendritic cells can engulf particulate Ags and induce T cell proliferative responses after pulsing with particulate Ag. However, their capacity to process viable Gram-negative bacteria for presentation by MHC-I and MHC-II has not been shown. We therefore characterized the ability of murine bone marrow-derived dendritic cells to process Escherichia coli and Salmonella typhimurium, expressing defined epitopes for presentation by MHC-I and MHC-II molecules. The I-Ak-restricted 46-61 epitope from hen egg white lysozyme (HEL(46-61)) or the Kb-restricted 257-264 epitope from chicken egg OVA (OVA(257-264)) was expressed as fusion proteins in the bacterial cytoplasm as the Crl-HEL and Crl-OVA fusion proteins, respectively. Bacteria expressing Crl-HEL or Crl-OVA, or beads coated with HEL or OVA, were coincubated with murine bone marrow-derived dendritic cells, and Ag processing and presentation were quantitated using T cell hybridomas. The data show that granulocyte-macrophage CSF-stimulated dendritic cells can process live intact Gram-negative bacteria for peptide presentation by MHC-I and MHC-II. Cytochalasin D inhibition studies revealed that processing for both MHC-I and MHC-II presentation required cytoskeletal rearrangements. Processing for MHC-I and MHC-II presentation was inhibited by ammonium chloride, suggesting that acidic compartments were required. Thus, granulocyte-macrophage CSF-stimulated murine bone marrow dendritic cells are capable of processing exogenous particulate Ags, including bacteria with no known mechanism for phagosomal escape, for peptide presentation by both MHC-I and MHC-II. These data suggest that dendritic cells may be important in priming both CD4+ and CD8+ T cells to bacterial Ags.