bluuebunny/crossref_metadata_2025 huggingface dataset:
B7-1 but not B7-2 efficiently costimulates CD8+ T lymphocytes in the P815 tumor system in vitro.
Authors: Gajewski T F
Published: January 2023

We recently have developed a method to generate primary P815-specific CTL in vitro from normal syngeneic splenocytes by employing transfection of B7-1 combined with exogenous IL-12 and IL-6. Surprisingly, when the homologous costimulator molecule B7-2 was substituted for B7-1 in this system, no specific CTL activity was obtained. Similarly, B7-1- but not B7-2-transfected P815 cells generated alloantigen-specific CTL activity from C57BL/6, accessory cell- and CD4(+)-depleted splenocytes, and costimulated proliferation of CD8+ lymphocytes in the presence of low doses of anti-CD3 mAb. In all systems, combined expression of both B7-1 and B7-2 costimulated as effectively as B7-1 alone, arguing against delivery of a dominant negative signal by B7-2. Proliferation of allogeneic, CD4(+)-depleted splenocytes in response to P815 cells, which relies on costimulation by normal accessory cells, was inhibited by anti-B7-1 but not anti-B7-2 mAbs. Finally, indirect evidence suggested a higher avidity of B7-1+ cells than B7-2+ cells for CTLA4. Thus, at least in the context of primary stimulation by irradiated P815 transfectants, B7-1 appears to be superior to B7-2 at costimulation of CD8+ T lymphocytes.
Reduction of early B lymphocyte precursors in transgenic mice overexpressing the murine heat-stable antigen.
Authors: Hough M R , Chappel M S , Sauvageau G , Takei F , Kay R , Humphries R K
Published: January 2023

To study the role of the murine heat-stable Ag (HSA) in lymphocyte maturation, we generated transgenic mice in which the HSA cDNA was under the transcriptional control of the TCR V beta promoter and Ig mu enhancer. The HSA transgene was expressed during all stages of B lymphocyte maturation. Expression was first detected in the earliest lymphoid-committed progenitors, which normally do not express HSA, and subsequently reached the highest levels in pro- and pre-B cells. In bone marrow, the number of IL-7-responsive clonogenic progenitors was < 4% of normal, whereas the frequency of earlier B lymphocyte-restricted precursors, detectable as Whitlock-Witte culture-initiating cells, was normal. Pro- and pre-B cells detected by flow cytometry were reduced by approximately 50% relative to controls. Mature splenic B cells were also reduced but to a lesser extent than in marrow, and their response to LPS stimulation was impaired. Reconstitution of SCID and BALB/c-nu/nu mice with HSA transgenic marrow indicated that the perturbations in B lymphopoiesis were not caused by a defective marrow microenvironment or by abnormal T cells. Our previous studies showed elevated HSA expression throughout thymocyte development, which resulted in a profound depletion of CD4+CD8+ double-positive and single-positive thymocytes. Together, these results indicate that HSA levels can determine the capacity of early T and B lymphoid progenitors to proliferate and survive. Therefore, HSA could serve as an important regulator during the early stages of B and T lymphopoiesis.
Selective activation-induced apoptosis of peripheral T cells imposed by macrophages. A potential mechanism of antigen-specific peripheral lymphocyte deletion.
Authors: Munn D H , Pressey J , Beall A C , Hudes R , Alderson M R
Published: January 2023

The self-reactive T cells that escape clonal deletion in the thymus must be suppressed by the less well characterized process of peripheral tolerance. In this study, we show that monocyte-derived macrophages (M phi) undergoing terminal differentiation in the presence of macrophage CSF (M-CSF) acquire the ability to selectively induce apoptosis of T cells in an activation-specific fashion. Lymphocytes were stimulated via the TCR using anti-CD3 cross-linking, staphylococcal superantigen, or allogeneic mixed-leukocyte cultures. T cells activated while in contact with M-CSF-derived M phi exited the resting G0 state and re-entered the cell cycle, but experienced a sustained arrest near the first G1/S transition, followed by progressive apoptosis. In contrast, lymphocytes that were not stimulated remained viable, and could later activate normally when removed from contact with M phi. Functionally, exposure of T cells to alloantigens presented by M-CSF-derived M phi resulted in a selective depletion of the alloresponsive T cell population, while preserving reactivity to other mitogens and to alloantigens from different donors. The ability of M phi to impose activation-induced apoptosis on lymphocytes was regulated developmentally, being absent in fresh monocytes, progressively acquired during differentiation in M-CSF, and abrogated if monocytes were exposed to IFN-gamma before differentiation. We speculate that this novel interaction may help to selectively delete autoreactive T cells that respond to self Ags presented by noninflammatory tissue M phi.
Ubiquitin-like moiety of the monoclonal nonspecific suppressor factor beta is responsible for its activity.
Authors: Nakamura M , Xavier R M , Tanigawa Y
Published: January 2023

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic Ag-nonspecific suppressive functions. Most recently, a cDNA clone encoding MNSF beta (a subunit of MNSF) was isolated and characterized. The MNSF beta cDNA encodes a 14.5-kDa fusion protein with MNSF-like activity consisting of an 8-kDa ubiquitin-like segment (ubi-L) and the ribosomal protein S30 (6.5 kDa). To determine whether ubi-L itself has biologic activity, cDNA encoding the ubi-L region was expressed in bacteria and the recombinant product was tested for the activity. Ubi-L showed MNSF-like biologic activity without any cytotoxic action. Interestingly, the addition of ubiquitin to the assay inhibited ubi-L-induced suppression. IFN-gamma, which is known to enhance the expression of MNSF receptor, induced splenocytes to secrete ubi-L by increasing mRNA. Ubi-L has species-specific action and the ability to selectively inhibit the B cell proliferative response stimulated by soluble but not by Sepharose-bound anti-Ig Ab. In addition, okadaic acid, a serine/threonine phosphatase 1 and 2A inhibitor, showed a synergistic inhibitory action with ubi-L, indicative of the possible involvement of phosphatase(s) in the regulation of ubi-L action.
Quantitative analysis of the expression of the HLA-DRB genes at the transcriptional level by competitive polymerase chain reaction.
Authors: Vincent R , Louis P , Gongora C , Papa I , Clot J , Eliaou J F
Published: January 2023

In addition to polymorphism of the peptide-binding site, the density of the MHC class II molecules expressed on the membrane of APC could well play a significant role in the MHC-peptide-TCR interaction during the immune response. We therefore investigated the regulation of the expression of the HLA-DRB genes at the transcriptional level. A competitive PCR approach was used to estimate the quantities of the HLA-DRB transcripts in peripheral blood B cells. When comparing the amounts of steady-state mRNAs among the different DRB1 alleles, the DRB1 transcripts in the DR52 haplotype group were found to be 2.5 to 3.5 times more abundant than the DRB1*01 transcripts, 1.5 to 2 times more abundant than the DRB1*04 transcripts, and 7 times more abundant than the DRB1*08 transcripts. Within the DR52 haplotype group, the DRB1 and DRB3 transcripts had the same abundance. Taken together, these results are in good agreement with the previously reported transcriptional activities of the DRB promoters except for DRB1*04, thus suggesting a differential post-transcriptional regulation among the DRB1 mRNAs.
Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific IFN-gamma response and increased production of T helper 2 cytokines.
Authors: Krishnan L , Guilbert L J , Russell A S , Wegmann T G , Mosmann T R , Belosevic M
Published: January 2023

Resolution of cutaneous leishmaniasis in infected mice is associated with a polarized Th1 immune response by the host, whereas maternal immune responses during pregnancy appear to be biased toward humoral (Th2) and away from cell-mediated (Th1) responses. The objective of this study was to evaluate whether the putative Th2 bias in pregnant C57BL/6 mice would impair their normal ability to mount a curative Th1 response against Leishmania major infection. Pregnant C57BL/6 mice developed larger cutaneous lesions that showed no signs of resolution up to 70 days after infection. The infection appeared to be contained but not cured, as the footpad lesion remained stable, neither decreasing (as in normal C57BL/6 mice) nor showing uncontrolled expansion leading to death (as in susceptible mouse strains such as BALB/c). The number of parasites harvested from the footpads of pregnant mice was markedly higher than controls throughout the course of infection. The increased severity of infection in pregnant mice was accompanied by reduced IFN-gamma and increased IL-4, IL-5, and IL-10 production by spleen and popliteal lymph node cells stimulated in vitro with Leishmania Ags. Furthermore, IgG1 was elevated in the serum of pregnant mice as opposed to an increase of IgG2a in infected but nonpregnant controls. These observations support the existence of a bias toward Th2 cytokine expression during pregnancy and suggest that these cytokines effectively down-regulate the course of a normal Th1 response against a parasite infection in the periphery.
Cellular detachment and deformation induce IL-8 gene expression in human bronchial epithelial cells.
Authors: Shibata Y , Nakamura H , Kato S , Tomoike H
Published: January 2023

Neutrophil elastase (NE) is known to be one of the most potent proteases capable of deforming and detaching human bronchial epithelial cells (BECs) and inducing IL-8 gene expression. However, mechanisms of NE-induced IL-8 gene expression are unclear, especially with respect to how they relate to cellular detachment. To elucidate these mechanisms, effects of cell detachment and deformation following mechanical injury or pharmacologic stimuli on IL-8 gene expression were examined by Northern analyses. When BET-1A cells from a human bronchial epithelial cell line were incubated with NE (100 nM), trypsin (0.5 mg/ml), EGTA (7 mM), or EDTA (0.7 mM) to induce deformation and detachment, IL-8 mRNA transcript levels were up-regulated, as demonstrated in a case of mechanical detachment from the culture plate using a cell scraper. This IL-8 gene expression was inhibited by pretreatment with 5 microM taxol, a microtubule-stabilizing agent. Colchicine or vinblastine, microtubule-disrupting agents, induced IL-8 gene expression, which was also inhibited by taxol treatment. These data suggest that structural changes, including deformation of the cytoskeleton, especially microtubules, may contribute to IL-8 gene expression in human BECs. Since detachment and cellular deformation of BECs caused by proteases have been observed frequently in a variety of inflammatory airway diseases, our findings provide evidence that detached or deformed BECs potentially enhance production of inflammatory mediators in the pathogenesis of airway inflammation.
Deficient CD4+ T cell proliferation in the class 1 MHC-restricted 2C TCR-transgenic mouse.
Authors: Chen F L , Kung J T
Published: January 2023

A comparative study of immune function and marker expression of CD4+ T cells from MHC class 1-restricted 2C TCR-transgenic (2C+) and control transgene-negative littermate (2C-) mice was performed. While 2C+CD4+ T cells resembled memory T cells on the basis of CD44highCD45RBlow expression, the majority of 2C-CD4+ T cells were of the CD44lowCD45RBhigh naive phenotype. Slightly lower levels of TCR-beta and CD3 were found on 2C+CD4+ T cell than 2C-CD4+ T cells. Vigorous proliferation by 2C-CD4+ T cells was observed upon stimulation with 1) anti-CD3 mAb presented through the FcR of macrophages; 2) immobilized (plate-bound) anti-CD3 + anti-CD28 mAbs; and 3) PMA + ionomycin. In marked contrast, all three mitogenic stimuli stimulated highly deficient proliferative responses by 2C+CD4+ T cells. However, significant IL-2 production was detected both in anti-CD3 and in PMA + ionomycin-stimulated cultures of 2C+CD4+ T cells. While intracellular calcium in 2C-CD4+ T cells rapidly increased following anti-CD3 addition, no such increase was observed for similarly stimulated 2C+CD4+ T cells. Anti-CD28, PMA, and coculture with 2C-CD4+ T cells each failed to significantly correct the deficient 2C+CD4+ T cells proliferation as induced by anti-CD3. In addition, IL-2, IL-4, and IL-7 supplements also failed to reverse the deficient proliferation of 2C+CD4+ T cells despite expression of IL-2R component alpha-, beta-chains and the gamma-chain common also to IL-4R and IL-7R. Thymus CD4+8- T cells from the 2C-transgenic mouse were similarly deficient in proliferation as spleen CD4+ T cells. A small subpopulation of CD4+ T cell from the 2C-transgenic mouse expressed the transgenic TCR alpha:beta heterodimer as detected by the 1B2 anti-2C clonotypic mAb; both 1B2+ and 1B2- subpopulations proliferated poorly in response to anti-CD3 and to PMA + ionomycin. These results raise the possibility that TCR engagement with MHC class 1 molecules during early intrathymic development can result in the emergence of CD4+ T cells characterized by unusual marker expression and function.
Evidence for the expression of HLAA-C class I mRNA and protein by human first trimester trophoblast.
Authors: King A , Boocock C , Sharkey A M , Gardner L , Beretta A , Siccardi A G , Loke Y W
Published: January 2023

In this study, evidence is provided that normal human first trimester extravillous trophoblast expresses class I HLA-C molecules in addition to HLA-G. cDNA from highly purified trophoblast cells obtained by flow cytometric sorting was amplified by reverse-transcriptase PCR using HLA locus-specific primers. The identity of the product was confirmed by Southern blotting and hybridization by a second HLA-C-specific oligonucleotide. HLA-C mRNA was clearly demonstrated in all trophoblast samples as well as in JEG-3 and BeWo choriocarcinoma cells. JAR choriocarcinoma cells did not express HLA-C. The presence of HLA-C protein in extravillous trophoblast was investigated using a panel of Abs: L31 is specific for heavy chains of all HLA-C alleles; Q1/28 reacts with all HLA class I products except HLA-G; HC-10 has preferential reactivity with HLA-B and HLA-C heavy chains. We performed 35S metabolic and 125I surface labeling of normal first trimester trophoblast and found abundant HLA-C intracellularly together with low levels of expression of both the beta 2m-associated forms and free heavy chains on the surface. Flow cytometric analysis of normal trophoblast confirmed the expression of a class I HLA molecule distinct from HLA-G by positive reactivity with Q1/28. Immunohistologic studies of first trimester placenta and the implantation site clearly showed expression of HLA-C in all extravillous trophoblast populations. Our results demonstrate the presence of two HLA class I molecules, HLA-G and HLA-C, on the surface of extravillous trophoblast. These results have implications in understanding how maternal uterine lymphocytes, notably the abundant NK-like cells, might recognize the implanting placenta.
Identification of MECL-1 (LMP-10) as the third IFN-gamma-inducible proteasome subunit.
Authors: Nandi D , Jiang H , Monaco J J
Published: January 2023

We show that six proteasome-associated proteins are induced by IFN-gamma, corresponding to three proteasome beta-type subunits and their precursors: the MHC-linked subunits (LMP-2 and LMP-7) and LMP-10. Concurrently, incorporation of LMP-9, LMP-17, and LMP-19 into proteasomes is reduced. LMP-10 appears to be the product of a previously cloned proteasome subunit gene, MECL-1. MECL-1 transcription is increased in the presence of IFN-gamma, whereas the transcription of two other proteasome genes, Lmp-15 and Lmp-3, is not affected. The three IFN-gamma-inducible subunits and their constitutively expressed counterparts contain most or all of the catalytic sites of the proteasome. Independent assortment of LMP-2, LMP-7, and LMP-10 into different proteasome complexes may thus generate up to 36 unique proteasome subsets. This may increase the repertoire of potentially antigenic peptides for presentation by MHC class I.
Bacterial superantigen-induced human lymphocyte responses are nitric oxide dependent and mediated by IL-12 and IFN-gamma.
Authors: Sriskandan S , Evans T J , Cohen J
Published: January 2023

Bacterial superantigens cause marked proliferation of T cells and release of lymphokines. Nitric oxide, derived from the conversion of L-arginine to L-citrulline, inhibits this activation in murine cells. We have now investigated the roles of IL-12, IFN-gamma, lymphotoxin-alpha, and nitric oxide during superantigen-induced human lymphocyte activation. Lymphocyte activation was determined by measurement of proliferative responses and lymphokine release. Both toxic shock syndrome toxin-1 from Staphylococcus aureus and recombinant streptococcal pyrogenic exotoxin A induced proliferation and production of IFN-gamma, lymphotoxin-alpha, and IL-12 by human mononuclear cells in a time-dependent fashion. The release of IFN-gamma was abrogated by a neutralizing Ab to IL-12, but lymphocyte proliferative responses were unaffected. A neutralizing Ab to IFN-gamma prevented the release of lymphotoxin-alpha, but did not affect proliferation. The neutralization of lymphotoxin-alpha using two different Abs did not affect IFN-gamma release or proliferation. In contrast to previous findings in mice, the arginine analogue, NG-monomethyl-L-arginine, significantly inhibited both proliferation and lymphokine release by superantigen-stimulated human cells. Thus, the release of lymphotoxin-alpha by lymphocytes following superantigen stimulation is dependent upon the presence of IFN-gamma; the IFN-gamma response is in turn under the control of IL-12. There is no evidence that nitric oxide plays an inhibitory role during superantigen-mediated human lymphocyte activation. Indeed, arginine is a prerequisite for such activation.
Effects of granulocyte-macrophage colony-stimulating factor on eicosanoid production by mononuclear phagocytes.
Authors: Brock T G , McNish R W , Coffey M J , Ojo T C , Phare S M , Peters-Golden M
Published: January 2023

Granulocyte-macrophage CSF (GM-CSF) primes granulocytes for leukotriene (LT) synthesis. Here, we examined the effects of GM-CSF on arachidonic acid (AA) metabolism in rat alveolar macrophages (AM), peritoneal macrophages, and human peripheral blood monocytes. Pretreatment of AMs with GM-CSF for 24 h significantly increased the synthesis of immunoreactive LTB4 upon subsequent stimulation with calcium ionophore. Enhanced LT synthesis required a minimum of 6 h of GM-CSF pretreatment, suggesting that protein synthesis was required for enhanced LT production; indeed, cycloheximide completely abolished the GM-CSF effect on LT synthesis. HPLC analysis confirmed that GM-CSF primed AMs for enhanced generation of LTB4, as well as the 5-lipoxygenase products LTC, and 5-HETE. Moreover, parallel increases in other AA metabolites and free AA were observed following GM-CSF pretreatment. The enhanced production of all AA metabolites indicated that GM-CSF up-regulated AA release. Consistent with this, whole cell lysates from GM-CSF-treated AMs demonstrated increased phospholipase A2 (PLA2) activity. The increased activity was resistant to DTT, indicating the involvement of a PLA2 other than the 14-kDa PLA2s. By immunoblot analysis, GM-CSF treatment caused an increase in the 85-kDa PLA2 protein comparable to the observed increase in PLA2 activity. Unlike AMs, neither peritoneal macrophages nor peripheral blood monocytes showed increased eicosanoid generation or increased expression of the 85-kDa PLA2 protein following GM-CSF pretreatment. These results indicate that GM-CSF increases the capacity of AMs, but not peritoneal macrophages or peripheral blood monocytes, to generate eicosanoids. This effect results from increased PLA2 activity, due at least in part to increased levels of the 85-kDa PLA2 protein.
TNF-alpha stimulates monocyte adhesion to glomerular mesangial cells. The role of intercellular adhesion molecule-1 gene expression and protein kinases.
Authors: Pai R , Bassa B , Kirschenbaum M A , Kamanna V S
Published: January 2023

TNF-alpha has been implicated in glomerular cell activation to produce adhesion molecules and monocyte chemoattractants associated with glomerular monocyte infiltration. This study examined the regulatory role of protein kinases and cAMP on TNF-alpha-induced intercellular adhesion molecules-1 (ICAM-1) expression and monocyte adhesion to mesangial cells. Activation of mesangial cells with TNF-alpha induced ICAM-1 mRNA and protein expression. Mesangial cells preincubated with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, stimulated both the gene and protein expression of ICAM-1. Mesangial cell PKC depletion abolished ICAM-1 mRNA message, while activation with TNF-alpha did not inhibit ICAM-1 transcripts. Preincubation of mesangial cells with calphostin C did not affect TNF-alpha-induced mesangial cell ICAM-1 message, while it blocked PMA-induced ICAM-1 mRNA expression. Protein tyrosine kinase (PTK) inhibitors blocked TNF-alpha-mediated mesangial cell ICAM-1 transcripts. cAMP-generating substances (e.g., pertussis toxin, isoproterenol, or dibutyryl cAMP) did not induce mesangial cell ICAM-1 gene expression. However, incubation of mesangial cells with TNF-alpha and dibutyrl cAMP blocked TNF-alpha-induced ICAM-1 message. Finally, preincubation of mesangial cells with TNF-alpha increased monocyte adhesion that could be blocked by anti-ICAM-1. Parallel to ICAM-1 gene expression data, TNF-alpha-induced monocyte-mesangial cell adhesion was inhibited by PTK inhibitors, but was not regulated through either PKC or intracellular cAMP-associated pathways. These results suggest that increased ICAM-1 expression by TNF-alpha activation of mesangial cells is one of the major pathways involved in monocyte adhesion to the mesangium, a phenomenon presumably regulated by signal-transduction pathways dependent on PTK, but not PKC or cAMP.
Monoclonal antibodies from NZW x BXSB F1 mice to beta2 glycoprotein I and cardiolipin. Species specificity and charge-dependent binding.
Authors: Monestier M , Kandiah D A , Kouts S , Novick K E , Ong G L , Radic M Z , Krilis S A
Published: January 2023

NZW x BXSB F1 mice develop a systemic autoimmune syndrome with various lupus-like manifestations. Male animals develop a degenerative coronary disease with myocardial infarction, resulting in death before 6 mo of age. The presence in these mice of anti-phospholipid Abs reacting with beta2-glycoprotein I may contribute to the pathogenesis of the cardiovascular lesions. beta2-glycoprotein I, a plasma protein implicated in various aspects of the coagulation pathway, is also the target of autoantibodies in humans with the anti-phospholipid syndrome. We obtained several mAbs from NZW x BXSB F1 mice that were selected for binding to cardiolipin. Two mAbs are specific for beta2-glycoprotein I and display a species-dependent pattern with preferential reactivity to mouse beta2-glycoprotein I. The other mAbs display charge-mediated interactions with anionic phospholipids in the absence of beta2-glycoprotein I. The analysis of the V region sequences of the mAbs suggests that cationic residues in the H chain complementarity-determining region 3 are important for their phospholipid reactivity. The structural features of the V(H)-D-J(H) junctions of these mAbs further support the view that an increased frequency of unusual V(D)J rearrangements directly contributes to the development of murine autoimmunity.
IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.
Authors: Willenborg D O , Fordham S , Bernard C C , Cowden W B , Ramshaw I A
Published: January 2023

129/Sv mice are resistant to induction of experimental autoimmune encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice of this strain lacking the gene coding for the ligand-binding chain of the IFN-gamma receptor develop EAE with high morbidity and mortality. Spleen cells from sensitized IFN-gammaR-/- mice proliferated extensively when stimulated with MOG peptide in culture and produced high levels of IFN-gamma and TNF but no detectable IL-4. Transfer of spleen cells from sensitized IFN-gammaR-/- mice produced EAE in both IFN-gammaR+/+ and IFN-gammaR-/- recipients. Disease was severe in IFN-gammaR-/- recipients and mortality high (77%). Surviving mice remained moribund until termination of the experiments. IFN-gammaR+/+ recipients developed disease of equal severity, but with no mortality, and recovered significantly. These results indicate that IFN-gamma is not essential for the generation or function of anti-MOG35-55 effector cells but does play an important role in down-regulating EAE at both the effector and induction phase of disease.
Enterotoxin septic shock protection and deficient T helper 2 cytokine production in growth hormone transgenic mice.
Authors: Gonzalo J A , Mazuchelli R , Mellado M , Frade J M , Carrera A C , von Kobbe C , Merida I , Martinez-A C
Published: January 2023

Neuroendocrine hormones have long been thought to play a role in lymphoid development and function. In particular, growth hormone has been shown to mediate thymic development as well as to promote T cell engraftment in severe combined immunodeficiency mice. Murine T helper cells are classified into two subsets based on their cytokine production pattern. Here, we report that transgenic mice for bovine growth hormone show significant alterations in T cell function and decreased capability for cytokine production, an effect that is more acute in T helper cells as measured by their inability to produce IL-4 upon in vivo injection with Staphylococcus aureus enterotoxin B. Furthermore, upon immunization with conventional Ags, growth hormone transgenic mice produce an altered Ig isotype pattern characterized by a response shift from IgG1 in nontransgenic mice to IgG2 in transgenic mice. The impaired T cell responses correlated with survival from septic shock mediated by bacterial enterotoxins. We conclude that growth hormone may have the potential of regulating immune responses in pathologic processes associated with hyperactivation of T cells or with massive cytokine production.
The xid mutation diminishes memory B cell generation but does not affect somatic hypermutation and selection.
Authors: Ridderstad A , Nossal G J , Tarlinton D M
Published: January 2023

In this study, we examine the relationship between primary and secondary T cell-dependent immune responses using the response of xid mice to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) as an experimental model. The reduced serologic primary immune response of xid mice was demonstrated to be caused by a substantially decreased Ab-forming cell (AFC) generation. Furthermore, the germinal center reaction in the primary xid immune response was diminished and the frequency of NP-specific memory B cells prior to secondary immunization was reduced 10-fold. Despite the poor primary response of xid mice, secondary exposure to Ag generated a response that was qualitatively and quantitatively equal to that of wt mice. The number of IgG1 AFCs in spleen and bone marrow increased equally in both groups, as did the proportion of AFCs secreting high affinity Ab in both locations. The extent and distribution of somatic mutations in the V(H) genes of xid secondary response B cells was also found to be normal, indicating that the xid gene product is not critical for the processes that result in affinity maturation. Thus, although xid mice generate memory B cells of normal phenotype but at a substantially lower frequency, this does not limit the magnitude of the secondary response. Therefore, our results imply that the reduced memory B cell frequency in xid mice is still above some threshold value necessary for a normal secondary immune response.
Postinduction transcriptional repression of E-selectin and vascular cell adhesion molecule-1.
Authors: Read M A , Neish A S , Gerritsen M E , Collins T
Published: January 2023

TNF-alpha induction of the E-selectin and vascular cell adhesion molecule-1 (VCAM-1) genes leads to transient accumulation of high levels of mRNA in endothelial cells. The increase in these mRNAs after induction is due to an increase in the rate of gene transcription, which is maintained for several hours in the continuous presence of cytokine. Cytokine-induced transcriptional activation of these genes requires the transcription factor, nuclear factor-kappaB. Following removal of TNF-alpha, there is rapid postinduction transcriptional repression common to both of these genes. The repression is protein synthesis dependent and correlates with protein synthesis-dependent loss of both the p50 and p65 subunits of nuclear factor-kappaB from the nucleus. IkappaBalpha is capable of specifically displacing endothelial-derived heterodimeric p50/p65 from the E-selectin and VCAM-1 kappaB elements, while having no effect on binding of p50 homodimer. In the presence of agents that block proteasomal degradation of IkappaBalpha, endogenous IkappaBalpha can be visualized in the nucleus of both resting and TNF-alpha-activated endothelial cells. Endogenous IkappaBalpha is readily detected in the nucleus of HeLa cells, and its nuclear localization is increased following removal of TNF-alpha. Repression of E-selectin and VCAM-1 transcription following cytokine removal requires the loss of nuclear p50 and p65, and involves IkappaBalpha. This postinduction transcription repression mechanism may be one component of a program that prevents inappropriate and prolonged expression of adhesion molecules.
Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1.
Authors: Halford W P , Gebhardt B M , Carr D J
Published: January 2023

Following ocular infection, herpes simplex virus type 1 (HSV-1) establishes latency in trigeminal ganglion (TG) neurons. Using reverse transcription-PCR, cytokine gene expression was analyzed in the TGs of mice infected with HSV-1. IL-2, TNF-alpha, IFN-gamma, IL-10, and RANTES mRNAs were readily detected in TGs taken from mice 7 days postinoculation (PI). Likewise, IL-2, IL-6, IL-10, and IFN-gamma protein were detected by ELISA of TG homogenates. Between 5 and 45 days PI, IL-10, IFN-gamma, TNF-alpha, and RANTES mRNAs were detected in nearly 100% of latently infected TGs (latent infection was confirmed by reverse transcription-PCR detection of HSV-1 latency-associated transcripts). T cell-associated cytokine and chemokine mRNAs (IL-2, IL-10, IFN-gamma, and RANTES) were still detected in the majority of latently infected TG samples taken between 60 and 135 days PI. In contrast, these cytokine mRNA species were rarely detected in uninfected TGs. Measurement of serum Abs to HSV-1 at different times revealed that anti-HSV-1 Ab concentrations approached a plateau in mice by 30 days PI but remained at high levels 67 and 125 days PI. Although there was molecular evidence of an ongoing immune response to HSV-1 in latently infected TG, histologic analysis indicated that very few mononuclear cells remained in the ganglion 60 days PI. Collectively, the results suggest that residual lymphocytes encounter viral Ag during HSV-1 latency with sufficient frequency to remain activated. The paradox of a persistent immune response against a latent infection is discussed.
Transcriptional up-regulation of intracellular adhesion molecule-1 in human endothelial cells by the antioxidant pyrrolidine dithiocarbamate involves the activation of activating protein-1.
Authors: Muñoz C , Castellanos M C , Alfranca A , Vara A , Esteban M A , Redondo J M , de Landázuri M O
Published: January 2023

The redox status of the cell plays an essential role in regulating signal transduction, transcription factor activity, and expression of cell surface molecules. In this study, we show that pyrrolidine dithiocarbamate (PDTC), a potent antioxidant agent, upregulated the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in human endothelial cells (EC). Further analysis of PDTC-mediated ICAM-1 up-regulation revealed that PDTC increased ICAM-1 mRNA levels and augmented its gene promoter activity. Transfection experiments in EC with reporter constructs harboring nested deletion fragments of the ICAM-1 promoter indicated the presence of a functional PDTC-responsive region located between positions -136 to -353 of the promoter. Gel retardation assays together with supershift analysis revealed that PDTC induced the binding of c-fos and c-jun to a consensus activating protein-1 (AP-1) binding site located at position -284. PDTC alone or in combination with TNF-alpha enhanced AP-1-dependent transactivation in HUVEC, as determined by DNA binding assays. The functional implication of AP-1 in the transcription of the ICAM-1 gene was further demonstrated by cotransfection experiments in which a c-jun expression vector induced the promoter activity of the PDTC-responsive element of the ICAM-1 promoter. Taken together, these results indicate that the antioxidant PDTC induces transcriptional activation of ICAM-1 and that this induction is mediated at least in part by the transcription factor AP-1. This mechanism might be operative in pathologic conditions in which a redox imbalance plays a key role, such as ischemia/reperfusion injury or arteriosclerosis.
Regulation of polymorphonuclear leukocyte cytokine receptor expression: the role of altered oxygen tensions and matrix proteins.
Authors: Simms H , D'Amico R
Published: January 2023

We investigated the effects of hypoxemia +/- reoxygenation (H/R) on matrix protein regulation of polymorphonuclear leukocyte (PMN) IL-1beta types I and II, TNF-alpha (p60, p80), and IL-8R expression compared with normoxic controls. H/R reduction IL-1beta type I, TNF-alpha (p80), and IL-8R expression compared with hypoxic levels. Neither fibronectin nor laminin effected cytokine receptor expression during normoxia, but both fibronectin and laminin significantly reduced IL-1beta type I, TNF-alpha (p80), and IL-8R expression during hypoxia. Following H/R, both fibronectin and laminin significantly reduced IL-1beta type I, TNF-alpha (p80), and IL-8R expression during hypoxia. Following H/R, both fibronectin and laminin significantly increased TNF-alpha (p60, p80) and IL-8R expression. Cross-linking of adherent PMN very late antigen (VLA)-5 and VLA-6 receptors resulted in a progressive increase in TNF-alpha (p60, p80) and IL-8R expression during hypoxia; cross-linking of adherent PMN VLA-5 and and VLA-6 receptors resulted in a progressive increase in TNF-alpha (p60, p80) and IL-8 receptors following H/R. Cross-linkage of IL-1betaR type I, TNF-alphaR (p80), and IL-8R during hypoxia and H/R resulted in increased and subsequently decreased O2- production and degranulation. Inhibition of reduced nicotinamide adenine dinucleotide phosphate oxidase activation with diphenyleneiodonium following hypoxia but before reoxygenation prevented the decreases in IL-1beta types I and II, TNF-alpha (p80), and IL-8R expression that was seen following H/R alone. These results demonstrate that, during hypoxia and H/R, integrin signaling via alpha5beta1, and alpha6beta1 increases and subsequently decreases the expression of PMN cytokine receptors.
Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene.
Authors: van Ginkel F W , McGhee J R , Liu C , Simecka J W , Yamamoto M , Frizzell R A , Sorscher E J , Kiyono H , Pascual D W
Published: January 2023

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.
Structural analysis of human antibodies to proteinase 3 from patients with Wegener granulomatosis.
Authors: Sibilia J , Benlagha K , Vanhille P , Ronco P , Brouet J C , Mariette X
Published: January 2023

We determined the structure of five IgM autoAbs to proteinase-3 (PR3). These Abs are highly specific for Wegener's granulomatosis (WG) and may be involved in the pathogenesis of vasculitis in WG. Five clonal lymphoblastoid cell lines secreting Abs to PR3 were derived from four patients' B cells. From 3 to 5% of supernatants from wells contained detectable anti-PR3 Abs, indicating that anti-neutrophil cytoplasmic Ab specificity represents a sizable part of the IgM B cell repertoire in patients with WG. Mu heavy chains of WG1, WG4-1, and WG4-2 clones belonged to the VH3 subgroup. WG4-1 and WG4-2 heavy chains were identical, indicating an oligoclonal expansion of autoreactive B cells in this patient. WG4-1 (and WG4-2) used the VH3-23 V(H) gene, the product of which was shown to directly bind PR3. Heavy chains of WG2 and WG3 derived from VH4-59 and VH1-2 genes, respectively. Comparison with germline sequences showed that three of the five V(H) genes from clonal lines were somatically mutated with a R:S ratio in complementarity-determining regions of 3:0, 5:1, and 5:1, respectively. Three kappa light chains derived from the Vkappa4 gene, and one derived from a Vkappa1 gene. In these four Vkappa genes, there were overall R:S ratios of mutation of 8:1 and 0:7 in complementarity-determining regions and framework regions, respectively. These data suggest that the production of these autoantibodies, which are increasingly important in the diagnosis and management of WG, are influenced by an Ag-driven process.
A cloned chicken lymphokine homologous to both mammalian IL-2 and IL-15.
Authors: Sundick R S , Gill-Dixon C
Published: January 2023

IL-2, cloned in several mammalian species, plays a critical role in immune system function. Indirect evidence suggests that IL-2-like molecules also exist in lower vertebrates, although none has been cloned. In view of the commercial importance of poultry and the lack of any IL-2 sequence data in lower vertebrates, we undertook to clone a chicken IL-2-like molecule. To this end, a cDNA library was constructed from activated chicken spleen cells and screened for T cell proliferative activity. Three clones of a single gene were isolated that produced a protein stimulatory for activated T cell blasts. This protein had 24 to 25% amino acid identity and 44 to 46% similarity to both bovine IL-2 and IL-15. The homology with IL-15 was unexpected, since primate IL-15 was reported to have no homology with IL-2. However, genetic distance analysis indicated that the chicken interleukin homology is closer to the mammalian IL-2 than to the mammalian IL-15 homology. Furthermore, the chicken gene has several characteristics of mammalian IL-2s: e.g., expression only by activated T cells, mRNA with a short 5' region preceding the open reading frame, and a short leader sequence. It has one characteristic that is unique to IL-15: four conserved cysteines allowing for two intrachain disulfide bonds.
The role of gammadelta T lymphocytes in lipopolysaccharide-induced eosinophil accumulation into the mouse pleural cavity.
Authors: Penido C , Castro-Faria-Neto H C , Larangeira A P , Rosas E C , Ribeiro-dos-Santos R , Bozza P T , Henriques M G
Published: January 2023

LPS induces an accumulation of eosinophils in the pleural cavity that requires resident macrophages and lymphocytes, but is independent of IL-5 production. In the present study we investigated the involvement of different T lymphocyte subsets on the modulation of LPS-induced eosinophil accumulation into the pleural cavity of mice. Within 4 h after LPS injection the number of neutrophils in the pleural cavity increased significantly. Mononuclear cell counts increased after 12 h, while a significant rise on eosinophil counts was observed only after 24 h. T lymphocytes counts were increased in the pleural cavity 24 and 48 h after LPS administration. This T lymphocyte accumulation was accounted for by an influx of the gammadelta+ subset, while CD4+ and CD8+ subsets did not accumulate in the pleural cavity after LPS stimulation. All those changes had resolved 96 h after LPS injection. Depletion of T lymphocytes by treatment with mAb anti-Thy 1.0 inhibited the eosinophil accumulation triggered by LPS. Aiming to clarify which T lymphocyte subset would be involved in the LPS-induced eosinophil accumulation, we depleted mice of various T lymphocyte subpopulations using specific Abs. Depletion of either CD4+ or CD8+ subsets failed to inhibit LPS-induced eosinophil migration. In contrast, when mice were treated with anti-gammadelta+ T lymphocyte mAb, a significant reduction of LPS-induced eosinophil accumulation was observed. Similarly, the administration of LPS in BALB/c-nu/nu mice induced the expected significant influx of eosinophils into the pleural cavity. Our results indicate that the gammadelta+ T lymphocytes are centrally involved in LPS-induced eosinophil accumulation in mice.
STAT5 involvement in the differentiation response of primary chicken myeloid progenitor cells to chicken myelomonocytic growth factor.
Authors: Woldman I , Mellitzer G , Kieslinger M , Buchhart D , Meinke A , Beug H , Decker T
Published: January 2023

We have investigated the activation of the STAT5 protein during the differentiation of myelomonocytic cells. In the human U937 promonocytic cell line, STAT5 activation occurred in response to several inducers of monocytic differentiation (phorbol ester, 1alpha,25-dihydroxyvitamin D3, and retinoic acid). In the promyelocytic HL60 cell line, STAT5 was activated in the course of either phorbol ester-induced monocytic differentiation or DMSO-induced granulocytic differentiation. To test for involvement of STAT5 in the differentiation of primary nonimmortalized cells, chicken myeloid progenitor cells transfected with the ts21-E26 avian retrovirus were studied. At 37 degrees C the temperature-sensitive oncoprotein of the ts21-E26 virus (p135(gag-myb-ets)) installs a differentiation block that is released by a shift to the nonpermissive temperature (42 degrees C). Both proliferation and differentiation of ts21-E26-transfected myeloblasts require the continuous presence of chicken myelomonocytic growth factor (cMGF). Addition of cMGF to growth factor-starved cells rapidly caused STAT5 tyrosine phosphorylation, DNA binding, and transcriptional activity at both permissive and nonpermissive temperatures. Moreover, the temperature shift-induced onset of myelomonocytic differentiation strictly correlated with the appearance of activated STAT5 in ts21-E26-infected myeloblasts, but not in cells infected with wtE26 retrovirus. These data position STAT5 in a nuclear signaling cascade induced by the cMGF receptor and suggest a contribution of STAT5 to the process of myelomonocytic differentiation or to the functional changes that accompany the maturation of myeloid progenitor cells to a terminally differentiated stage.
Suppressive effects of recombinant human monokine induced by IFN-gamma (rHuMig) chemokine on the number of committed and primitive hemopoietic progenitors in liquid cultures of CD34+ human bone marrow cells.
Authors: Schwartz G N , Liao F , Gress R E , Farber J M
Published: January 2023

Studies in this report investigated potential hemopoietic suppressive effects of human monokine induced by IFN-gamma (HuMig), a CXC chemokine that is chemotactic for activated lymphocytes. rHuMig was purified from Trichoplusia ni cells after infection with a recombinant baculovirus. The recombinant protein was added to liquid cultures of CD34+ human marrow cells stimulated with IL-3 alone or with both IL-3 and either insulin-like growth factor II (IGF-II) or stem cell growth factor (SCF). The number of committed progenitors, colony-forming units for granulocytes and macrophages (CFU-GM), and primitive progenitors, long term culture-initiating cells (LTC-IC) derived from liquid cultures of CD34+ cells, was determined. rHuMig abrogated the IGF-II-dependent enhancement of CFU-GM and long term culture-initiating cell numbers. Additional studies demonstrated that in liquid cultures of CD34+ cells both rHuMig and IFN-inducible protein-10, another CXC chemokine that is related to HuMig, inhibited the production or expansion of CFU-GM. For a subset of marrows, rHuMig also abrogated SCF enhancement of CFU-GM numbers in cultures of CD34+ cells stimulated with both IL-3 and SCF. These studies are the first to demonstrate that rHuMig can act as a negative regulator of in vitro hemopoiesis, that both rHuMig and IP-10 can suppress an increase in the number of committed progenitors from CD34+ cells, and that chemokines can abrogate hemopoietic stimulatory effects of IGF-II.
Effect of an ectokinase inhibitor, K252b, on degranulation and Ca2+ signals of RBL-2H3 cells and human basophils.
Authors: Teshima R , Saito Y , Ikebuchi H , Rajiva De Silva N , Morita Y , Nakanishi M , Sawada J , Kitani S
Published: January 2023

We examined the effects of K252b, an ectoprotein kinase inhibitor of microbial origin, on the activation process of RBL-2H3 cells by cross-linking of IgE receptors by the endoplasmic reticulum Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone or by the Ca2+ ionophore A23187. Analysis of phosphorylation of ectoproteins following IgE receptor cross-linking revealed that K252b mainly inhibited the phosphorylation of a 130-kDa protein. The inhibitor simultaneously inhibited degranulation and the sustained increase in the cytosolic calcium ion concentration even after addition of Ag. In contrast, K252b did not inhibit the increase in degranulation and cytosolic calcium ion concentration caused by stimulation with 2,5-di(tert-butyl)-1,4-hydroquinone and A23187. Permeation of K252b into RBL-2H3 cells, assessed by fluorescence intensity, was very low. K252b also inhibited degranulation caused by IgE receptor cross-linking in human basophils, but did not inhibit the degranulation caused by A23187. Thus, our findings suggest that the effects of K252b may be mediated by outer surface-bound or -anchored K252b-sensitive molecules on RBL-2H3 cells and human basophils, and that the phosphorylation of ectoprotein may involve a transmembrane influx of Ca2+ by IgE receptor cross-linking.