bluuebunny/crossref_metadata_2025 huggingface dataset:
Voltage-dependent and calcium-activated ion channels in the human mast cell line HMC-1
Authors: Duffy S M , Leyland M L , Conley E C , Bradding P
Published: September 2022

The mechanisms underlying the recruitment, differentiation, and sustained activation of mast cells in disease are likely to include modulation of ion channels. Specific Ca2+, K+, and Cl− conductances have been identified in rodent mast cells, but there are no equivalent data on human mast cells. We have used the whole-cell patch-clamp technique to characterize macroscopic ion currents in both the human mast cell line HMC-1 and human skin mast cells (HSMCs) at rest and in HMC-1 after activation with calcium ionophore. HSMCs were electrically silent at rest. In contrast, HMC-1 expressed a strong outwardly rectifying voltage-dependent Cl− conductance characteristic of ClC-4 or ClC-5 and a small inwardly rectifying K+ current not carried by the classical Kir family of K+ channels. Calcium ionophore induced the appearance of outwardly rectifying Ca2+-activated Cl− and K+ currents, while hypotonicity induced another outwardly rectifying conductance typical of ClC-3. Reverse transcription-PCRs confirmed that mRNAs for the voltage-dependent Cl− channels ClC-3 and –5 were expressed. This is the first definitive description of a ClC-4/5-like current in a native leukocyte. We suggest that this current may contribute to the malignant phenotype while the Ca2+-activated K+ and Cl− currents may be involved in cell activation.
Monocyte differentiation in intestine-like macrophage phenotype induced by epithelial cells
Authors: Spöttl T , Hausmann M , Kreutz M , Peuker A , Vogl D , Schölmerich J , Falk W , Andreesen R , Andus T , Herfarth H , Rogler G
Published: September 2022

Macrophages in normal colonic mucosa show a specific and distinct phenotype with low expression of the typical monocyte/macrophage surface antigens CD14, CD16, and CD11b and T-cell costimulatory molecules. A method for the in vitro induction of a macrophage phenotype similar to this intestinal phenotype is presented. Multicellular spheroids (MCSs) of intestinal epithelial cell (IEC) and control cell lines were cocultured with elutriated monocytes. Surface antigen expression was analyzed by immunohistochemistry and flow cytometry. Interleukin (IL)-1β mRNA was measured by quantitative PCR. Monocytes adhered and infiltrated the MCSs within 24 h. In the MCSs of all IEC lines, the typical monocyte/macrophage surface antigens CD14, CD16, CD11b, and CD11c, which are detectable after 24 h of coculture by immunohistochemistry and flow cytometry, were down-regulated after 7 days (e.g., for CD14 at 24 h, expression was 86% of CD33+ cells; at day 7, it was 11%). A clear decrease of lipopolysaccharide (LPS)-stimulated IL-1β transcription in monocytes cocultured with IEC MCSs could be observed during the 7-day period. For the first time an intestine-like macrophage-phenotype could be induced in vitro. Interactions with IECs play an essential role during this differentiation, which is of functional relevance, e.g., for LPS-induced cytokine secretion.
Feedback activation of T-cell antigen-presenting cells during interactions with T-cell responders
Authors: Mannie Mark D , Walker Mindi R
Published: September 2022

Like many T cells in the myelin basic protein (MBP)-specific T-cell repertoire, CD4− GP2.3H3.16 (3H3) T cells recognize guinea pig MBP as an agonist but recognize autologous rat (R)MBP as a mixed agonist/antagonist. 3H3 T cells do not exhibit proliferative responses to RMBP but nonetheless respond to RMBP by accumulation of T-cell surface I-A/peptide complexes and generation of T-cell antigen-presenting cell (T-APC) activity. This study showed that presentation of RMBP by 3H3 T-APC is long-lived but is lost during interactions with cognate responders or on overt activation of T-APCs. Presentation of RMBP to encephalitogenic T cells resulted in the reciprocal activation of 3H3 T-APCs as evidenced by blastogenesis, proliferation, and induction of interleukin-2R and OX40 markers on 3H3 T-APC. These data indicate that T-APCs, like B-cell APCs, undergo clonal expansion after presentation of a cognate antigen to T-cell responders.
Deleterious effects ofEchinacea purpureaand melatonin on myeloid cells in mouse spleen and bone marrow
Authors: Currier N L , Sicotte M , Miller S C
Published: September 2022

The neurohormone, melatonin, a product of the pineal gland, is a potent immune cell stimulant. Phytochemicals contained in root extracts of the plant species Echinacea purpurea are also potent as immune cell stimulants. Both agents are potent stimulants of T, B, and/or natural killer cells, but little is known of their effect on other hemopoietic cells, specifically granular leukocytes, also participants in a wide variety of disease defense processes. Given their current popularity and availability for amelioration of a) jet lag and sleep disorders (melatonin) and b) virus-mediated respiratory infections (E. purpurea), we investigated the effects of these agents on granular leukocytes and their precursors, myeloid cells. Mice received these agents daily for 7 or 14 days via the diet, thus mimicking human administration, after which spleens and bone marrow were removed and assessed for mature, differentiated granulocytes and their myeloid progenitors. The influence of these agents was directly related to the stage of cell maturity. Administration of both agents together resulted in significantly elevated levels of myeloid progenitor cells in both bone marrow and spleen and significantly reduced levels of mature, functional granulocyte progeny in both organs, suggesting a) increased precursor proliferation, b) antiapoptosis among the progenitors, and/or c) inhibition of precursor maturation—the latter readily explaining the paucity of mature granulocyte progeny. In conclusion, individual administration of either the herbal derivative and melatonin was either without effect (E. purpurea) or even advantageous (melatonin) to cells of this lineage, but when administered together, these agents significantly perturbed myelopoiesis.
Functional HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by HIV strain X4
Authors: Tchou I , Misery L , Sabido O , Dezutter-Dambuyant C , Bourlet T , Moja P , Hamzeh H , Peguet-Navarro J , Schmitt D , Genin C
Published: September 2022

HIV can cross the intact epithelium of genital mucosae via Langerhans cells. Fresh Langerhans cells are known to express CD4 and CCR5. The presence of CXCR4 on the surface of cultured but not freshly isolated Langerhans cells has been described. In the present study, we demonstrate that CXCR4 was expressed by fresh Langerhans cells isolated and purified from epidermis. However, the percentage of Langerhans cells expressing CXCR4 or CCR5 increased during maturation of the cells in culture, especially in the presence of exogenous granulocyte-macrophage colony-stimulating factor. To determine whether CXCR4 was functional, freshly isolated Langerhans cells were infected with HIV LAI, a T-cell-tropic strain, and p24 protein production was measured in culture supernatants. p24 production was observed when infected Langerhans cells were cocultured with SupT1 cells. However, the presence of HIV provirus DNA was evidenced within the infected Langerhans cells by nested PCR. Ultrastructural studies confirmed the formation of syncytia when Langerhans cells were cocultured with SupT1 cells. Preincubation of Langerhans cells with azidothymidine or SDF-1-α, a natural ligand for CXCR4, prevented infection. These data demonstrated that CXCR4 is present on the surface of Langerhans cells freshly isolated from human skin epidermis and that this expression is functional.
Muramyl dipeptide and mononuclear cell supernatant induce Langhans-type cells from human monocytes
Authors: Mizuno Kana , Okamoto Hiroyuki , Horio Takeshi
Published: September 2022

Muramyl dipeptide (MDP) in bacterial cell walls reportedly evokes epithelioid cell granulomas. We examined its effects on multinucleated-giant-cell (MGC) formation from monocytes. Supernatant of concanavalin A-stimulated peripheral blood mononuclear cells (conditioned medium) generated MGCs from monocytes. MDP significantly increased the fusion index of Langhans-type MGCs (LGCs) but did not affect total MGCs. N-Acetylmuramyl-l-alanyl-l-isoglutamine, an MDP analogue, had no effect on MGC formation. MGCs were produced by conditioned medium from CD14++/CD16− monocytes. MDP enhanced the LGC fusion index from CD14++/CD16− monocytes. MGCs were not produced from CD14+/CD16+ monocytes or immature dendritic cells induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL) 4 and only weakly produced from macrophage (M)-CSF- or GM-CSF-induced macrophages. Added MDP did not generate MGCs from CD14+/CD16+ monocytes or dendritic cells but enhanced LGC formation from macrophages. Because IFN-γ, IL-3, and GM-CSF reportedly are important in LGC induction, we added anti-IFN-γ, anti-IL-3, or anti-GM-CSF monoclonal antibody (mAb) concomitantly to the monocyte culture treated with conditioned medium alone or plus MDP. Anti-IFN-γ mAb completely abrogated MGC generation, whereas anti-GM-CSF and anti-IL-3 mAbs significantly inhibited LGCs. These findings suggest that CD14++/CD16− monocytes are fused to form LGCs by MDP derived from granulomatous-disease-causing pathogens with inflammatory mediators such as IFN-γ, IL-3, and GM-CSF.
Agonist-dependent failure of neutrophil function in diabetes correlates with extent of hyperglycemia
Authors: McManus Linda M , Bloodworth Rebecca C , Prihoda Thomas J , Blodgett Janet L , Pinckard R Neal
Published: September 2022

Inexplicable controversies with regard to possible functional defects of neutrophilic polymorphonuclear leukocytes (PMNs) in diabetes persist. The purpose of the present study was to elucidate the relative effectiveness of several PMN agonists in stimulating lysosomal-enzyme secretion and leukotriene (LT) B4 production by PMNs isolated from diabetic subjects. Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induced significantly less lysosomal-enzyme secretion and LTB4 production in diabetic-subject PMNs than in normal-subject PMNs. It is surprising that PMNs from these same diabetic subjects responded normally after stimulation with A23187, serum-opsonized zymosan, or phorbol myristate acetate. The in vitro responsiveness of PMNs stimulated with fMLP or PAF was inversely correlated with indices of in vivo glycemic control (fasting plasma glucose and glycated-hemoglobin levels). In combination, these results indicate that hyperglycemia is associated with sustained decreases in PMN function but only in response to agonists that initiate stimulus-response coupling via G-protein-coupled receptors. This agonist-selective reduction in PMN responsiveness may contribute to the compromised host defense associated with sustained hyperglycemia in diabetes.
Induction of fibroblast-like cells from CD34+ progenitor cells of the bone marrow in rheumatoid arthritis
Authors: Hirohata Shunsei , Yanagida Tamiko , Nagai Tatsuo , Sawada Tetsuji , Nakamura Hiroshi , Yoshino Shin'ichi , Tomita Tetsuya , Ochi Takahiro
Published: September 2022

To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34+ cells from bone marrow to generate fibroblast-like type B synoviocytes. CD34+ cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast-like morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor α (TNF-α), much more effectively than CD34+ cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast-like cells was not at all observed in cultures with SCF, GM-CSF, and interleukin 4 (IL-4) with or without TNF-α. Generation of fibroblast-like cells was correlated with matrix metalloproteinase (MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were significantly higher in TNF-α-stimulated cultures of bone marrow CD34+ cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34+ cells from patients with RA have abnormal capacities to respond to TNF-α and to differentiate into fibroblast-like cells producing MMP-1, suggesting that bone marrow CD34+ progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.
IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression
Authors: Mukai Takao , Iwasaki Masayuki , Gao Ping , Tomura Michio , Yashiro-Ohtani Yumi , Ono Shiro , Murai Masako , Matsushima Kouji , Kurimoto Masashi , Kogo Mikihiko , Matsuya Tokuzo , Fujiwara Hiromi , Hamaoka Toshiyuki
Published: September 2022

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)-12 or IL-2 alone (designated 2D6IL-12 or 2D6IL-2, respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up-regulating the function of adhesion molecules. 2D6IL-12 grew, forming cell aggregates, in culture containing IL-12. This was due to lymphocyte function-associated antigen (LFA)-1–intercellular adhesion molecule (ICAM)-1 interaction, because 2D6IL-12 expressed both LFA-1 and ICAM-1 and cell aggregation was inhibited by anti-ICAM-1 monoclonal antibody. Despite comparable levels of LFA-1 and ICAM-1 expression, 2D6IL-2 cells did not aggregate in culture with IL-2. It is important that there was a critical difference in CCR5 expression between 2D6IL-12 and 2D6IL-2; the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL-12 or IL-2, IL-18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL-12 and IL-18 strikingly up-regulated 2D6 cell aggregation. Notably, LFA-1-mediated aggregation of 2D6IL-12 cells was suppressed by anti-CCR5 antibody. These results indicate that IL-12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5-mediated activation of LFA-1 molecules.
CXCR4 undergoes complex lineage and inducing agent-dependent dissociation of expression and functional responsiveness to SDF-1α during myeloid differentiation
Authors: Gupta Shalley K , Pillarisetti Kodandaram , Aiyar Nambi
Published: September 2022

The CXC chemokine SDF-1 and its receptor CXCR4 mediate myelopoiesis, presumably by regulating the homing of hematopoietic progenitor cells. We used the inducible HL-60 cell line as a model system for comparative analysis of CXCR4 expression during differential maturation into the granulocytic or monocytic phenotypes. Five different measures of CXCR4 expression and functional coupling: mRNA and surface expression, SDF-1-mediated [35S]GTPγS binding, calcium flux, and chemotaxis were examined simultaneously. Granulocytic differentiation with dimethyl sulfoxide induced surface expression of CXCR4 as well as SDF-1-mediated [35S]GTPγS binding and chemotaxis, whereas calcium flux was attenuated by twofold to threefold in HL-60 cells. Conversely, monocytic differentiation with vitamin D3 inhibited surface expression and SDF-1-mediated chemotaxis, even as it induced [35S]GTPγS binding and calcium flux by more than twofold. Sodium butyrate up-regulated all parameters of CXCR4 expression studied. Together, these results demonstrate that CXCR4 expression undergoes complex regulation at multiple checkpoints, with the likely involvement of different G-proteins for signal transduction during cellular differentiation and following activation with SDF-1.
Crotalus durissus terrificussnake venom regulates macrophage metabolism and function
Authors: Sampaio S C , Sousa-e-Silva M C C , Borelli P , Curi R , Cury Y
Published: September 2022

In the present study, we examined the effect of Crotalus durissus terrificus venom on rat macrophage metabolism and function. Two hours after subcutaneous injection of the venom, peritoneal resident (unstimulated), elicited (thioglycollate-stimulated), and activatedMycobacterium bovis strain bacille Calmette Guérin (BCG) macrophages were collected, and their functional and metabolic parameters were analyzed. The venom inhibited spreading and phagocytosis of macrophages. On the other hand, this treatment increased H2O2 and NO production, candidacidal activity, and the activities of key enzymes of glycolysis and glutaminolysis. We also investigated whether the venom could affect macrophage activation by thioglycollate or BCG. The administration of venom 2 h before injection of thioglycollate and BCG or 2 or 3 days after injection of the thioglycollate or BCG, respectively, did not modify the previous observations. These findings suggest that crotalic venom leads the macrophage to an activated state, with high production of oxygen- and nitrogen-reactive species. This cell activation state does not include inflammatory properties of spreading and phagocytosis.
Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis
Authors: Cao Jay , Bobo Jeffrey A , Liuzzi Juan P , Cousins Robert J
Published: September 2022

Zinc is critical for the functional and structural integrity of cells. We have used the monocytic cell line THP-1 as a model in which to study both the responsiveness of metallothionein and ZIP2 transporter expression to zinc depletion induced by the intracellular zinc chelator TPEN [N, N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine] and the extent of concomitant apoptosis. Metallothionein expression increased proportionately with the addition of zinc to the medium and decreased with TPEN treatment. When treated with TPEN, both THP-1 cells and human peripheral blood mononuclear cells exhibited marked decreases in cellular zinc concentrations and increases in ZIP2 mRNA expression. These results suggest that cells attempt to homeostatically adjust to zinc depletion. When THP-1 cells were treated with >5 μM TPEN, cell viability decreased, and cells entered the early stages of apoptosis. These data show that metallothionein and ZIP2 expression are inversely related during zinc depletion and that apoptosis is concurrent with these changes.
IL-10 receptor dysfunction in macrophages during chronic inflammation
Authors: Avdiushko Rita , Hongo David , Lake-Bullock Helene , Kaplan Alan , Cohen Donald
Published: September 2022

The immunosuppressive activity of interleukin-10 (IL-10) makes this cytokine a potentially important clinical tool to reduce inflammatory responses in various diseases. Its efficacy as a therapeutic modality is dependent on the responsiveness of immune cells. We report that macrophages from mice chronically infected with the LP-BM5 retrovirus had a reduced capacity to respond to IL-10 in vitro. The ability of IL-10 to inhibit lipopolysaccharide-induced production of tumor necrosis factor (TNF) α and IL-6 was significantly reduced in both alveolar and peritoneal macrophages from infected versus uninfected mice. IL-10 hyporesponsiveness was not related to direct infection by the retrovirus, because bone marrow-derived macrophages infected in vitro with LP-BM5 were as responsive to IL-10 as were uninfected bone marrow-derived macrophages. TNF-α appeared to contribute to development of IL-10 hyporesponsiveness, because exposure of normal macrophages to TNF-α but not interferon-γ reduced macrophage responsiveness to IL-10. Reverse transcriptase-PCR and flow cytometry demonstrated normal expression of the α and β chains of the IL-10 receptor in macrophages from infected mice, suggesting that IL-10 hyporesponsiveness is not related to a change in receptor expression. The potential role of reduced IL-10 responsiveness in the chronicity of inflammation in this and other diseases is discussed.
Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages
Authors: Misra Uma K , Adlakha Charu L , Gawdi Govind , McMillian Michael K , Pizzo Salvatore V , Laskowitz Daniel T
Published: September 2022

Apolipoprotein E (ApoE) is a 34-kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca2+ stores. This cascade is inhibited by pretreatment with receptor-associated protein and Ni2+, and it is mediated by a pertussis toxin-sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor-related protein. A peptide derived from the receptor-binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE-mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE-dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.
Increased nitric oxide production by airway cells of sensitized and challenged IL-10 knockout mice
Authors: Ameredes Bill T , Zamora Ruben , Gibson Kevin F , Billiar Timothy R , Dixon-McCarthy Barbara , Watkins Simon , Calhoun William J
Published: September 2022

The anti-inflammatory cytokine interleukin (IL)-10 suppresses induciblenitric oxide synthase (iNOS); therefore, NO production should increasein the absence of IL-10. Production of NO (as nitrite) bybronchoalveolar lavage cells of IL-10 knockout (−/−) micewas assessed after ovalbumin sensitization and airway challenge (S/C)and was compared with the IL-10-sufficient, wild-type (WT) C57Bl6.Eosinophil recruitment occurred in S/C WT and IL-10−/−mice, suggesting allergic airway inflammation. Alveolar macrophages(per g mouse) were unchanged (∼3×104 cells) with theexception of a doubling in the S/C IL-10−/− mice(∼6×104 cells, P<0.05). NO production (permillion cells) was doubled in cells from S/C IL-10−/−(15.3 μM) mice compared with WT (7.6 μM, P<0.05). Inhibition of iNOS byl-N5-(1-iminoethyl)-ornithine reduced NOproduction in all S/C mice, confirming that the increase was a resultof up-regulation of iNOS. We conclude that IL-10 is a critical cytokineregulating iNOS in murine airway cells and that its absence can lead toup-regulation of iNOS and development of allergic airwayinflammation.
Profound effect of the absence of IL-4 on T cell responses during infection withSchistosoma mansoni
Authors: Pedras-Vasconcelos João A , Brunet Laura Rosa , Pearce Edward J
Published: September 2022

T cell responses of interleukin (IL)-4−/− and wild-type (WT) mice infected with the helper T cell 2 (Th2) response-inducing pathogen Schistosoma mansoni were compared. As expected, given the important role of IL-4 in Th2 response induction, the absence of IL-4 resulted in diminished Th2 responses, apparent as reduced production of IL-4, -5, and -10 by CD4+ cells isolated from the spleens of infected IL-4−/− mice. Surprisingly, these cells produced significantly less interferon (IFN)-γ and proliferated less than did those from infected WT mice after T cell receptor ligation. CD8+ cells isolated from infected IL-4−/− mice also produced less IFN-γ than WT CD8 cells, although there was no difference in the proliferative responses of these cell populations. After infection, spleens of infected IL-4−/− mice did not enlarge to the same extent as those of WT mice, and attrition of the CD8+ cell population within this lymphoid organ was noted. Taken together, the data indicate that in addition to inhibiting Th2 response development, the lack of IL-4 during schistosomiasis significantly affects additional aspects of T cell responses.
Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-α
Authors: Gorak-Stolinska Patricia , Truman Jean-Philip , Kemeny David M , Noble Alistair
Published: September 2022

Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-α/TNFR interactions because although type 1 cells secreted more TNF-α than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.
Effect of cross-tolerance between endotoxin and TNF-α or IL-1β on cellular signaling and mediator production
Authors: Ferlito Marcella , Romanenko Olga G , Ashton Sarah , Squadrito Francesco , Halushka Perry V , Cook James A
Published: September 2022

Abstract: Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) α and interleukin (IL)-1β. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1β, and TNF-α stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor κBα (IκBα) DNA binding of nuclear factor-κB (NF-κB), and mediator production were examined. In naive cells, LPS, TNF-α, and IL-1β induced IκBα degradation, kinase phosphorylation, and NF-κB DNA binding. LPS stimulation induced production of TNF-α or TxB2 and degradation of IRAK. However, neither TNF-α nor IL-1β induced IRAK degradation or stimulated TNF-α or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-α production. LPS pretreatment induced cross-tolerance to TNF-α or IL-1β stimulation. Pretreatment with TNF-α induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1β did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-α and TxB2 production. These data demonstrate that IL-1β induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-κB activation.
Regulation of nuclear factor κB activation by G-protein-coupled receptors
Authors: Ye Richard D
Published: September 2022

Accumulating evidence indicates that G-protein-coupled receptors (GPCRs) play an active role in transcriptional regulation. In leukocytes, activation of receptors for several chemokines and classic chemoattractants has been associated with enhanced expression of proinflammatory cytokines and chemokines. GPCRs in endothelial and epithelial cells also regulate transcription and contribute to the expression of cytokines, adhesion molecules, and growth factors that are essential for extravasation of leukocytes and tissue repair. Nuclear factor (NF) κB is one of the most important transcription factors responsible for the expression of these proinflammatory genes. Recent studies have shown that GPCRs utilize several different pathways to activate NF-κB. These pathways differ from the ones induced by classic cytokines in that they are initiated by heterotrimeric G-proteins, but they converge to IκB phosphorylation and nuclear translocation/modification of the NF-κB proteins. GPCR-induced NF-κB activation provides an effective means for local expression of cytokine and growth factor genes due to the wide distribution of these receptors. Chemokine-induced, GPCR-mediated production of chemokines constitutes an autocrine regulatory mechanism for the growth of certain malignant tumors and enhances the recruitment of leukocytes to sites of inflammation.
Estrogen restores cellular immunity in injured male mice via suppression of interleukin-6 production
Authors: Messingham Kelly A N , Heinrich Scott A , Kovacs Elizabeth J
Published: September 2022

This study examined whether estrogen treatment can improve immunity in male mice after combined ethanol and burn injuries. 17β-Estradiol [estrogen, given subcutaneously (s.c.)] or oil (control) was administered at 30 min and 24 h postinjury. At 48 h postinjury, ethanol/burn-injured mice demonstrated significant suppression of cellular immunity. Estrogen treatment restored the delayed-type hypersensitivity (P<0.01) and splenocyte-proliferative (P<0.05) responses, reduced macrophage interleukin-6 (IL-6) (P<0.05), and increased survival after bacterial challenge (P<0.01). In vitro neutralization of IL-6, combined with macrophage supernatant experiments, confirmed that the beneficial effects of estrogen treatment were mediated through modulation of macrophage IL-6 production. Moreover, estrogen treatment resulted in a decrease in splenic nuclear factor-κB (NF-κB) activation in injured mice. There were no changes in cellular NF-κB or IκBα protein expression or IκBα phosphorylation at serine 32. Taken together, these studies suggest that estrogen treatment of injured male mice improves cellular immunity through direct modulation of NF-κB activation.
cAMP-elevating agents suppress dendritic cell function
Authors: Kambayashi Taku , Wallin Robert P A , Ljunggren Hans-Gustaf
Published: September 2022

The administration of cAMP-elevating agents affects a number of autoimmune and inflammatory conditions. Because dendritic cells (DCs) play a pivotal role in autoimmunity and inflammation, the isolated effects of cAMP-elevating agents on the function of DCs was examined. In a dose-dependent manner, 8-Bromo cAMP, prostaglandin E2, and 3-isobutyl-1-methylxanthine inhibited tumor necrosis factor α release and suppressed antigen presentation by DCs. The same effect was observed with rolipram, a specific inhibitor of phosphodiesterase type 4, but not with inhibitors of other phosphodiesterases. The decreased antigen presentation by DCs was associated with an enhanced production of interleukin (IL)-10 and with lower major histocompatibility complex type II (MHC II) expression. Furthermore, the inhibition of antigen presentation and MHC II expression was significantly reversed by treatment of DCs with neutralizing antibody against IL-10, suggesting the involvement of an IL-10-dependent mechanism. Taken together, these results might explain why certain cAMP-elevating agents such as rolipram are effective in blocking autoimmunity and inflammation.
T-cell-conditioned medium efficiently induces the maturation and function of human dendritic cells
Authors: Kato Kazunori , Takaue Yoichi , Wakasugi Hiro
Published: September 2022

We present evidence that T-cell-conditioned media (TCCM) can efficiently induce human immature dendritic cells (DC) to express high levels of immune accessory molecules commonly found on mature DC. TCCM prepared from cell-free supernatants of anti-CD3-activated T cells contained several soluble factors including CD40-ligand (sCD40L), TNF-α, and IFN-γ. In contrast to moderate up-regulation of costimulatory molecules by the addition of individual cytokines or monocyte-conditioned medium, treatment of immature DC with TCCM induced a marked increase in the expression of costimulatory molecules in a dose-dependent manner. The ability of TCCM to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for CD40L, TNF-α, and IFN-γ, indicating that these factors present in TCCM are mainly implicated in the maturation of DC. Importantly, TCCM-treated DC can produce significantly higher levels of IL-12 and are highly effective stimulators in allogenenic and autologous mixed-lymphocyte reactions. Overall, these findings show that cultivation with TCCM is an efficient approach for the induction of mature DC that should be useful in eliciting antigen-specific immune responses against cancer and viruses.
Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells
Authors: Liu Ru , Aupperle Karl , Terkeltaub Robert
Published: September 2022

Neutrophil-dependent inflammation dependent on monosodium urate (MSU)crystal-induced IL-8 expression occurs in gout. MSU crystals activatephagocyte Src family tyrosine kinases and the serine/threonine kinasep70s6k. Thus, using monocytic THP-1 cells, we assessed the potentialfor Src family kinases and p70s6k to mediate MSU-induced IL-8expression. MSU crystals induced phosphorylation of p70s6k and the Srckinases c-Src, Lyn, Hck, and Fyn. IL-8 expression was attenuated moreby the Src kinase inhibitor PP1 than by the p70s6k inhibitor rapamycin.PP1 inhibited crystal-induced phosphorylation of ERK1/2 and IκBαand suppressed IκB kinase (IKK) activation and NF-κB binding to theIL-8 promoter, signals that mediate MSU-induced IL-8 expression.Transfection of the native Src inhibitor, C-terminal Src kinase (Csk),also suppressed crystal-induced c-Src, ERK1/2, and IκBαphosphorylation and IL-8 expression. We conclude that Src familytyrosine kinase signaling plays a significant role in MSUcrystal-induced IL-8 expression via stimulation of ERK1/2 pathway andNF-κB activation.
Shiga toxin 1-induced activation of c-Jun NH2-terminal kinase and p38 in the human monocytic cell line THP-1: possible involvement in the production of TNF-α
Authors: Foster Gregory H , Tesh Vernon L
Published: September 2022

Shiga toxin-producing enterohemorrhagic E. coli infections cause bloody diarrhea, which may progress to life-threatening complications such as the hemolytic-uremic syndrome (HUS). HUS patients frequently have elevated levels of the proinflammatory cytokine tumor necrosis factor α (TNF-α) detectable in urine. Thus, sequelae may develop following the localized production of proinflammatory cytokines within the kidneys. A possible source of these cytokines are macrophages, which respond to the toxins by producing TNF-α. We have shown previously that THP-1 cells produce soluble TNF-α in response to the toxins, whose production requires host-cell tyrosine-kinase activity and toxin-enzymatic activity. To further examine signaling pathways involved in TNF-α expression, we determined that JNK1 and -2 and p38, but not ERK1 or -2, were phosphorylated following toxin exposure. Blockade of p38 activation reduced TNF-α production following Shiga toxin 1 treatment. Finally, we present a model of the ribotoxic stress response triggered in human macrophages by Shiga toxins.
Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning
Authors: Nijhuis Evert , Lammers Jan-Willem J , Koenderman Leo , Coffer Paul J
Published: September 2022

Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.
Activation of extracellular signal-related kinase by TNF-α controls the maturation and function of murine dendritic cells
Authors: Yanagawa Yoshiki , Iijima Norifumi , Iwabuchi Kazuya , Onoé Kazunori
Published: September 2022

Functional roles of extracellular signal-related kinase (ERK)activation in dendritic-cell (DC) maturation have been unclear. In thepresent study, we investigated the ERK pathway in tumor necrosis factor(TNF)-α-induced maturation of murine spleen-derived DC. TNF-αincreased surface expressions of major histocompatibility(MHC) and costimulatory molecules on DC in a dose-dependentmanner. High (40 ng/ml) and low (0.4 ng/ml) concentrations of TNF-αmarkedly enhanced ERK1/2 activation in DC, and this activation wasblocked completely by PD98059, a selective inhibitor of the ERKpathway. When DC were treated with TNF-α at a low but not a highconcentration, PD98059 notably enhanced surface expressions of the MHCand costimulatory molecules and allostimulatory capability of the DC. Interleukin (IL)-12 production was enhanced significantly by PD98059 in DC treated with low or high concentration of TNF-α. These findingssuggest that TNF-α-induced ERK activation negatively controlsmaturation and IL-12 production in murine DC.
NF-κB-mediated transcriptional regulation of human β-defensin-2 gene following lipopolysaccharide stimulation
Authors: Tsutsumi-Ishii Yuko , Nagaoka Isao
Published: September 2022

β-Defensins are cationic peptides with broad-spectrum antimicrobialactivities that contribute to innate host defense. Among humanβ-defensins (hBDs), hBD-2 is up-regulated in epithelial tissues andmononuclear phagocytes in response to bacterial infection andproinflammatory cytokines. However, little is known about the molecularmechanism of hBD-2 gene regulation. Here, we investigatedlipopolysaccharide (LPS)-mediated transcriptional regulation of thehBD-2 gene by focusing on the roles of NF-κB, STAT, and NF-IL-6 sitesin mononuclear phagocytes using RAW264.7 cells, which are sensitive to LPS. Luciferase reporter analyses demonstrated that two NF-κB siteswere essential for full LPS responsiveness of the hBD-2 gene. Further, both NF-κB sites were also crucial for basal transcriptionalactivity. In contrast, neither the NF-IL-6 nor STAT binding site wasrequired for LPS-induced hBD-2 transcription. Electrophoretic mobilityshift assay indicated that in unstimulated cells, NF-κB p50 homodimerbound to both NF-κB sites, whereas the p65-p50 heterodimer formedcomplexes with these sites following LPS stimulation. Together, theseobservations indicate that NF-κB plays an important role in theregulation of hBD-2 gene expression in response to LPS.
AP-1 is essential for p67phoxpromoter activity
Authors: Gauss Katherine A , Bunger Peggy L , Quinn Mark T
Published: September 2022

The cytosolic NADPH oxidase cofactor p67phoxhas been shown to be one of the limiting factors in assembly andactivation of this multi-protein enzyme complex and, therefore, must behighly regulated at the transcriptional level. In the present studies, we have further characterized the promoter for humanp67phox. Genomic sequence upstream of thetranslational start site (TLS; 2 kb) was cloned, and RACE was used toidentify and compare the transcriptional start site (TSS) in twomyeloid cell lines, HL-60 and PLB-985. Two major TSS were identifiedwithin the first intron for both cell lines, and one transcriptisolated from PLB-985 cells started approximately 34 bp 5′ of exon 1and contained no intron 1 sequence. To identify regulatory regions ofthe promoter, a luciferase reporter was used to assay a series of promoter deletion constructs. The greatest transcriptional activity wasobserved for fragments containing at least 500 bp upstream of the TLS. Sequence analysis of the p67phox promoterrevealed consensus binding sites for previously described transcriptionfactors including AP-1 and PU.1. Site-directed mutagenesis of the AP-1site demonstrated that this site was essential for basal transcription. EMSA, competition, and super-shift assays showed that this site wasspecifically recognized by nuclear factors of the AP-1 family. EMSAanalysis and promoter-reporter assays with the PU.1 consensus sites atpositons -176, -283, and -328 demonstrate that PU.1 binds the site atposition -283 with high affinity. Mutagenesis of any one of the PU.1sites reduced the basal transcriptional activity by approximately 50%,demonstrating that, although none of these sites is singularlyresponsible for the basal transcriptional activity, all three sitesplay some role in the transcriptional activity of thep67phox promoter. In support of thisconclusion, mutagenesis of all three sites completely abrogatedtranscriptional activity.
Expression and functional characterization of CFTR in mast cells
Authors: Kulka M , Gilchrist M , Duszyk M , Befus A D
Published: September 2022

Mast cell activation requires Cl− flux, which maintains the driving force for entry of extracellular calcium and initiates release of mediators such as histamine. However, chloride channel expression in mast cells has been poorly understood. For the first time, reverse transcriptase-polymerase chain reaction shows that rat-cultured mast cells (RCMC) and peritoneal mast cells (PMC) contain mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR), an important chloride channel. Immunostaining with an anti-CFTR antibody indicates expression of CFTR in PMC and RCMC. Mast cell CFTR is a functional Cl− channel because it is capable of mediating Cl− flux in response to elevated cAMP. An inhibitor of CFTR-dependent Cl− flux, diphenylamine-2-carboxylate down-regulates mast cell mediator release. These results show that rat mast cells express a functional CFTR, which might be important in mediator release.
Mycobacterium aviuminfection of macrophages results in progressive suppression of interleukin-12 production in vitro and in vivo
Authors: Wagner Dirk , Sangari Felix J , Kim Sang , Petrofsky Mary , Bermudez Luiz E
Published: September 2022

Interleukin-12 (IL-12) has been shown to have an important role in thehost defense against Mycobacterium avium. We sought todetermine if human monocyte-derived macrophages produce IL-12 uponM. avium infection. Although IL-12 can be measured insupernatants of M. avium-infected macrophages at 24, 48,and 72 h following infection, intracellular staining showed that24 to 48 h after infection, IL-12 was synthesized chiefly byuninfected macrophages in the monolayer, suggesting that M.avium infection inhibits IL-12 production. In addition, the dataalso suggest that the longer macrophage monolayers were infected, theless IL-12 they were able to produce. Stimulation of macrophages withIFN-γ prior to infection with M. avium resulted ingreater production of IL-12 compared with unstimulated macrophages. Culture supernatant of M. avium-infected macrophagemonolayers, but not control macrophages, partially inhibited IL-12production by IFN-γ-stimulated macrophages. This partial inhibitionwas not reversed by antiinterleukin-10 (anti-IL-10) andantitransforming growth factor β1 (anti-TGFβ1)-neutralizingantibodies. M. avium infection of macrophages in vitro alsosuppressed IL-12 synthesis induced by Listeriamonocytogenes infection. Immunohistochemistry staining of spleenof infected mice showed that IL-12 production by splenic macrophageswas more pronounced in the beginning of the infection but decreasedlater. Our data indicate that M. avium infection of macrophages suppresses IL-12 production by infected cells and that thesuppression was not a result of the presence of IL-10 and TGFβ1 inthe culture supernatant.
Regulation of gelatinase B in human monocytic and endothelial cells by PECAM-1 ligation and its modulation by interferon-beta
Authors: Nelissen Inge , Ronsse Isabelle , Van Damme Jo , Opdenakker Ghislain
Published: September 2022

Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) andgelatinase B are coexpressed at sites of inflammation, where an intenseinteraction occurs between leukocytes and endothelial cells. Toinvestigate whether a functional link exists between PECAM-1 activationand gelatinase B production, the regulatory role of PECAM-1, IFN-γ,IFN-β, LPS, and PMA on the production of gelatinase B (MMP-9) wasstudied in vitro in normal human umbilical vein endothelial cells(HUVECs), human peripheral blood mononuclear cells (PBMCs), and in ahuman monocytic leukemia cell line. In THP-1 cells, progelatinase Blevels were slightly up-regulated by immobilized PECAM-1-specificmonoclonal antibody (mAb) and soluble recombinant PECAM-1 when comparedwith strong induction by LPS and PMA. IFN-β inhibited the induced andbasal gelatinase B production but had no modulating effect on theexpression of PECAM-1. HUVECs mainly produced progelatinase A(proMMP-2). Treatment with LPS and triggering of the endothelial cellswith PECAM-1 mAb or recombinant PECAM-1 had no effect on gelatinase Aor B production, whereas PMA stimulated the production of progelatinaseB. IFN-β significantly up-regulated the expression of PECAM-1 in HUVECs but did not affect gelatinase secretion. Finally, in PBMCs, progelatinase B production was increased by soluble PECAM-1 mAb, recombinant PECAM-1, LPS, and PMA, whereas IFN-β reduced gelatinase Bsecretion. IFN-β did not alter PECAM-1 expression on PBMCs. Thus, PECAM-1 and gelatinase B are differently regulated in leukocytes andendothelial cells.
Cross-linking of GPI-80, a possible regulatory molecule of cell adhesion, induces up-regulation of CD11b/CD18 expression on neutrophil surfaces and shedding of L-selectin
Authors: Yoshitake Hiroshi , Takeda Yuji , Nitto Takeaki , Sendo Fujiro
Published: September 2022

Previously, we described a novel glycosylphosphatidyl inositol (GPI)-anchored glycoprotein (designated GPI-80) on human neutrophils and monocytes that may regulate β2 integrin-dependent neutrophil adherence and migration. However, the mechanism regulating β2 integrin remains to be clarified. To study this, we examined changes in β2 integrin expression and function caused by cross-linking GPI-80. GPI-80 cross-linking induced up-regulation of CD11b/CD18 (Mac-1) expression on neutrophil surfaces and shedding of L-selectin, which depends on tyrosine phosphorylation and cytoskeleton remodeling. Furthermore, the cross-linking enhanced fMLP-induced human neutrophil adherence. These results suggest that GPI-80 may be a regulator of β2 integrin in neutrophils.
Bacterial metabolite interference with maturation of human monocyte-derived dendritic cells
Authors: Säemann Marcus D , Parolini Ornella , Böhmig Georg A , Kelemen Peter , Krieger Peter-Michael , Neumüller Josef , Knarr Katharina , Kammlander Willibald , Hörl Walter H , Diakos Christos , Stuhlmeier Karl , Zlabinger Gerhard J
Published: September 2022

Dendritic cells (DC), the most potent APC, are central to antimicrobial immunity. Because of evolutionary pressure, it is reasonable that pathogens have evolved strategies to also subvert this host-defense mechanism. In the present study, we describe a novel way of bacterial interference with DC maturation. The bacterial metaboliten-butyrate, which occurs physiologically in high concentrations in the gastrointestinal tract and has well-known anti-inflammatory effects, is able to prevent LPS-induced maturation of DC resulting in a reduced capability to stimulate T cells. In particular, n-butyrate prevents homotypic DC clustering, inhibits IL-12 while sparing IL-10 production, and at the molecular level, blocks NF-κB translocation. These results demonstrate efficient targeting of DC function by a bacterial metabolite, which might explain the particular type of immune responsiveness in the presence of this bacterial agent as exemplified in the gastrointestinal tract.