bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-alpha inhibits antigen-induced eosinophil and CD4+ T cell recruitment into tissue.
Authors: Nakajima H , Nakao A , Watanabe Y , Yoshida S , Iwamoto I
Published: December 2022

In a murine model of airway late phase reaction, we have previously shown that Ag-induced eosinophil recruitment into the tissue of sensitized mice is mediated by CD4+ T cells and IL-5. To determine whether IFN-alpha regulates Ag-induced eosinophil recruitment into tissue, we studied the effects of rIFN-alpha, endogenous IFN-alpha production induced by poly I.C, and anti-IFN-alpha mAb on the eosinophil infiltration of the trachea induced by Ag inhalation in mice. The i.p. administration of rIFN-alpha prevented Ag-induced eosinophil infiltration in the trachea of sensitized mice. In addition, the inhibitory effect of rIFN-alpha on Ag-induced eosinophil infiltration was not recovered by pretreatment with anti-IFN-gamma mAb. The administration of rIFN-alpha also inhibited Ag-induced CD4+ T cell infiltration in the trachea. However, the administration of rIFN-alpha did not significantly affect blood eosinophil counts nor the number of blood and splenic CD4+ T cells. On the other hand, pretreatment with anti-murine IFN-alpha mAb enhanced Ag-induced eosinophil and CD4+ T cell infiltration in the trachea. Finally, endogenous IFN-alpha/beta production induced by poly I.C also inhibited Ag-induced eosinophil infiltration in the trachea and this inhibition of the eosinophil infiltration was abrogated by pretreatment with anti-IFN-alpha/beta mAb. These results indicate that IFN-alpha suppresses Ag-induced eosinophil and CD4+ T cell recruitment into tissue.
Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa.
Authors: Porges A J , Redecha P B , Kimberly W T , Csernok E , Gross W L , Kimberly R P
Published: December 2022

The presence of anti-neutrophil cytoplasmic Abs (ANCA) in many patients with systemic vasculitis suggests that ANCA may play a role in disease pathogenesis. Neutrophils from patients with Wegener's granulomatosis often express ANCA target Ags (myeloperoxidase (MPO) and proteinase 3 (PR3)) on their surface, making these intracellular primary granule enzymes accessible to these autoantibodies. Similarly, normal neutrophils can be induced to translocate MPO and PR3 to the cell surface in vitro, and we demonstrate that murine mAb ANCA IgG, but not IgM, binds to the ANCA target and engages the Fc gamma RIIa ligand-binding site on the surface of human neutrophils. In contrast to ANCA IgM, ANCA IgG also induces an oxidative burst in neutrophils (oxidation of dihydrorhodamine = 91 +/- 15 fluorescence units with anti-PR3 IgG vs 17 +/- 2 with anti-PR3 IgM, p < 0.001). Blockade of the ligand-binding site of Fc gamma RIIa with an antibinding site mAb Fab significantly reduces this ANCA IgG-triggered production of reactive oxygen species (p < 0.01). Similarly, human ANCA bind the ANCA target, engage Fc gamma RIIa, and induce an oxidative burst in neutrophils. The allelic phenotype of Fc gamma RIIa strongly influences the Fc gamma receptor engagement by ligand, and Fc gamma RIIa homozygous donors differ by more than threefold in the quantitative production of reactive oxygen intermediates (ROI) (p < 0.01). Thus, engagement of Fc gamma RIIa by the Fc region of ANCA is one mechanism by which these autoantibodies activate receptor-mediated signal transduction systems in human neutrophils to initiate programs of inflammation and tissue injury. Fc gamma receptor alleles may represent heritable disease risk factors influencing the magnitude of such a process.
Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFc gamma RIII-A) and the associated Fc epsilon RI gamma-chain.
Authors: Radeke H H , Gessner J E , Uciechowski P , Mägert H J , Schmidt R E , Resch K
Published: December 2022

Immune complexes localize to the glomerular mesangium in many forms of human glomerulonephritis. In this investigation, we determined whether primary cultured human glomerular mesangial cells (HMC) express mRNA and functional protein of FcR for IgG. Performing reverse transcription-PCR with subsequent control Southern blots, we showed that in contrast to human monocytes or granulocytes, mRNA for Fc gamma RI and Fc gamma RII is not detectable in either growth-arrested (48 h serum free) or unstimulated proliferating (medium with 10% FCS) HMC. However, IFN-gamma in combination with LPSE.coli (LPS), but not LPS alone elicited a significant transcription of hFc gamma RIII mRNA in resting HMC, whereas cytokines, such as IL-1 beta or TGF-beta, failed to induce any of the three Fc gamma Rs in resting HMC. Proliferating HMC showed a basal transcription of Fc gamma RIII mRNA, which was enhanced by IFN-gamma in combination with LPS. Slot-blot analysis indicated that only the Fc gamma RIII-A gene encoding the transmembrane isoform of Fc gamma RIII was expressed by HMC. For the first time, transcripts for the gamma-chain of Fc epsilon RI were found in HMC. Fc gamma RIII protein expression was detected after LPS/IFN-gamma stimulation both by specific staining of paraformaldehyde-fixed HMC and immunoprecipitation of Fc gamma RIII protein from HMC membranes. Fc gamma RIII-A receptors are functionally active, as HMC IL-6 mRNA synthesis was stimulated by heat-aggregated IgG or F(ab')2 fragments of CLBGran1 mAb only after induction of Fc gamma RIII-A. These in vitro data suggest that HMC that are basically negative for Fc gamma RIII can be stimulated to express low affinity Fc gamma Rs, and thus may participate actively in human glomerulonephritis involving immune complexes.
Inhibition of macrophage Ia expression by nitric oxide.
Authors: Sicher S C , Vazquez M A , Lu C Y
Published: December 2022

Bacterial LPS inhibits the expression of Ia by macrophages stimulated by IFN-gamma. We now present the following observations that suggest a causal relationship between nitric oxide (NO) and this inhibition of Ia expression: 1) NO production precedes inhibition of Ia, 2) the ability of LPS to inhibit Ia expression by IFN-gamma stimulated macrophages is correlated in a dose-dependent fashion with NO production, 3) Ia expression is restored if NO production is inhibited by NG-monomethyl-L-arginine or culturing the macrophages in L-arginine-free medium, and 4) exogenous NO inhibits IFN-gamma-stimulated Ia expression. Taken together these experiments indicate that NO inhibits macrophage expression of Ia. Furthermore, the following studies showed that inhibition of Ia by NO was not due to macrophage death: trypan blue exclusion, macrophage adhesion, conversion of the tetrazolium dye (MTT) to its formazan by a functioning electron transport system, and phagocytosis of IgG opsonized SRBCs. By inhibiting Ia expression, NO may inhibit Ag-presentation to T cells, secretion of IFN-gamma by these T cells, and ultimately inhibit the IFN-gamma-dependent production of NO synthetase. This inhibitory mechanism may prevent excessive NO formation and tissue injury.
Role of CD27 in T cell immune response. Analysis by recombinant soluble CD27.
Authors: Agematsu K , Kobata T , Sugita K , Freeman G J , Beckmann M P , Schlossman S F , Morimoto C
Published: December 2022

CD27 is a disulfide-linked 120-kDa transmembrane glycoprotein expressed on the majority of T cells, B cells, and NK cells; it has homology to a family of molecules that includes the receptors for nerve growth factor and TNF. Previous studies strongly suggest that the CD27 molecule plays a key role in the process of T cell activation. To further determine its role in T cell activation, a recombinant soluble molecule composing only the extracellular domain of CD27 was produced by transfection of Chinese hamster ovary cells. We have defined the binding properties of recombinant soluble CD27 (rsCD27) to CD27 ligand (CD27L) cDNA transfected NIH 3T3 cells and have determined its functional effects on in vitro T cell activation as well as on PWM-driven B cell IgG synthesis. rsCD27 bound specifically to CD27L and the binding was inhibited by one of our anti-CD27 mAbs, anti-1A4, suggesting that the 1A4 epitope of CD27 plays a role in the binding to CD27L. Functionally, rsCD27 inhibited T cell proliferation induced by various stimuli, such as PHA, tetanus toxoid, and anti-CD2, as well as PWM-driven B cell IgG synthesis, similar to the effects of adding anti-1A4. Determination of CD27L expression showed that CD27L mRNA is induced rapidly on activated T and B cells. Taken together, these results provide direct evidence that CD27-CD27L interaction plays a critical role in T cell activation as well as in T cell-dependent B cell IgG synthesis, suggesting that the CD27-CD27L interaction may constitute a component of the T cell-T cell or T cell-B cell interaction seen after activation with Ag or mitogen.
Cellular mechanisms that maintain neonatally-induced tolerance of class II alloantigens. Evidence that factor-mediated suppression silences cytotoxic T cell activity.
Authors: Matriano J A , Socarras S , Streilein J W
Published: December 2022

Virtually all neonatal mice of the A strain background are rendered profoundly and permanently tolerant of test skin allografts if they receive an i.v. inoculation of semiallogeneic hematopoietic cells expressing class II disparate alloantigens. After neonatally injected mice reach immunologic maturity, their lymphoid organs have been found to contain 1) tolerogen-specific CD4+ T cells that proliferate and secrete IL-4 when stimulated in vitro with class II tolerogens and 2) tolerogen-specific CD8+ T cell that fail to differentiate into cytotoxic effector cells. In this study, experiments are described that investigate the possibility that tolerance is maintained by regulatory T cells of the Th2-type. When A.TH T cells were stimulated in vitro with A.TL alloantigens in the presence of lymphoid cells from tolerant mice, tolerogen-specific cytotoxic T cells responses were absent or greatly diminished. When regulatory cells from tolerant mice were fractionated and tested, the cell type responsible for suppression proved to be CD4+ class II tolerogen-specific and gamma irradiation sensitive. Moreover, suppression of tolerogen-specific cytotoxic T cell generation was achieved when regulatory cells and naive responder cells were separated by a transwell barrier and supernatants harvested from cultures in which tolerant cells were stimulated specifically with class II tolerogens also inhibited cytotoxic T cell generation. Thus, suppression appears to be mediated by a soluble factor(s) produced by regulatory T cells. We conclude that tolerance of class II alloantigens is maintained, at least in part, by regulatory cells of the Th2-type that secrete factors that suppress the generation of tolerogen-specific effector cells required for rejection of solid tissue allografts.
Transmission of an Mls-1a-like superantigen to BALB/c mice by foster-nursing on F1 Mls-1bxa mothers. Sex-influenced onset of clonal deletion.
Authors: Piazzon I , Goldman A , Torello S , Nepomnaschy I , Deroche A , Dran G
Published: December 2022

Foster nursing of BALB/c (Mls-1b) mice on (BALB/cxAKR/J)F1 and (BALB/cxDBA/2)F1 (Mls-1bxa), but not on (BALB/cxC57Bl/6)F1 or (BALB/cxC3H/He)F1 (Mls-1bxb mothers, induced the progressive deletion of V beta 6+ and V beta 8.1+ T cells in 50% of the litter. The onset of this Mls-1a-like clonal deletion was markedly sex-influenced, being earlier in females (8-10 wk of age) than in males (32 wk). In both sexes, CD4+ V beta 6+ cells were more affected than CD8+ V beta 6+ cells. Decreases in the percentage of V beta 6+ cells were detected simultaneously in the thymus, lymph nodes, and peripheral blood. Preceding clonal deletion, functional unresponsiveness of CD4+ T cells to Mls-1 a Ags and to anti-V beta 6 Abs could be detected in most young male and female mice. The transmission of the Mls-1a-like superantigen through foster-nursing on (BALB/cxAKR/J)F1 mice correlated with the presence in milk of the mouse mammary tumor virus envelope protein gp52.
Antigen-specific early primary humoral responses modulate immunodominance of B cell epitopes.
Authors: Vijayakrishnan L , Kumar V , Agrewala J N , Mishra G C , Rao K V
Published: December 2022

Accumulation of primary Abs against two distinct epitopes on a designed polypeptide, MEP-1, was examined. The early primary response was predominantly against a C-terminal epitope (MEP 77-100), although subsequent maturation established an epitope within the N-terminal half (MEP 17-31) as the immunodominant one. Inversion of immunodominance correlated with the inability of anti-MEP 77-100 B cells to interact productively with T cells, which consequently received reduced help. Interaction between epitope-specific B and T cells was found to be attenuated in the presence of early primary anti-MEP-1 antiserum, and the extent of inhibition was directly proportional to the level and affinity of epitope-specific Igs. Therefore, it seems that early primary Abs to a multideterminant Ag selectively down-regulate maturation of epitope-specific primary humoral responses.
Identification of amino acids within the MHC molecule important for the interaction with the adenovirus protein E3/19K.
Authors: Feuerbach D , Etteldorf S , Ebenau-Jehle C , Abastado J P , Madden D , Burgert H G
Published: December 2022

The E3/19K protein of human adenovirus type 2 binds to class I MHC Ags thereby interfering with their cell surface expression and Ag presentation function. Currently, it is unclear exactly which structure of MHC molecules is recognized by the E3/19K protein. We have previously demonstrated that the murine H-2Kd Ag is able to associate with E3/19K, whereas the allelic H-2Kk molecule is not. By using exon shuffling between Kd and Kk molecules, the alpha 1 and alpha 2 domains of MHC class I molecules were identified as essential structures for binding the viral protein. In this report, we have examined the contribution of individual amino acids within the alpha 2 domain of MHC for binding E3/19K. First, we show that within this domain the alpha-helical part is most important for the interaction with E3/19K. By using site-directed mutagenesis, Kd-specific amino acids were introduced into the alpha-helix of the alpha 2 domain of Kk. By using the expression of mutagenized proteins in E3/19K+ cells, we have identified Tyr 156 and Leu 180 as being essential for the association with the E3/19K protein. In addition, Kd residue Glu 163 seems to contribute to the complex formation. Furthermore, analysis of a panel of Kd/Dd recombinants indicates that a similar region in the Dd molecule, namely, the C-terminal half of the alpha 2 domain, affects binding to E3/19K. Combining these results with Ab binding data, we present two alternative models of how the adenovirus protein may bind to the alpha 1 and alpha 2 domains.
A novel population of expanded human CD3+CD56+ cells derived from T cells with potent in vivo antitumor activity in mice with severe combined immunodeficiency.
Authors: Lu P H , Negrin R S
Published: December 2022

Recently, we have reported a novel protocol for the generation of highly efficient cytotoxic effector cells by culturing PBLs in the presence of IFN-gamma, IL-2, mAb against CD3, and IL-1 alpha. We have termed these cultures cytokine-induced killer (CIK) cells because the phenotype of the cells with the greatest cytotoxicity expresses both the T cell marker CD3 and the NK cell marker CD56. Cells with this phenotype are rare (approximately 1 to approximately 5%) in uncultured PBLs. CD3+CD56+ cells expand nearly 1000-fold under these culture conditions. The majority of the CD3+CD56+ cytotoxic cells in CIK cultures were derived from CD3+CD56- T cells, and not CD3-CD56+ NK cells. Expression of CD56, but not CD8, on CD3+ cells correlated with the greatest cytotoxicity against various cellular targets. We have used mice with severe combined immunodeficiency (SCID) injected with human lymphoma cells to evaluate the in vivo antitumor effects of CIK vs lymphokine-activated killer (LAK) cells. Groups of animals inoculated with 1 x 10(6) SU-DHL4 cells (a human B lymphoma cell line with a t(14;18) chromosomal translocation), injected 1 day later with CIK cells either i.v. or i.p., had significantly prolonged survival compared with control animals injected with tumor cells alone (median survival 90 days vs 58 days, p < 0.001) or animals treated with LAK cells (median survival 90 days vs 68 days, p < 0.002). Approximately 30% of the SCID mice challenged with SU-DHL4 cells and treated with CIK cells became long-term survivors compared with none of the animals treated with LAK cells. No molecular evidence of occult lymphoma was found in the CIK cell-treated long-term survivors when their bone marrow, spleen, liver, and lung were analyzed by t(14;18) PCR at the end of 6 mo. By using these culture conditions, a novel population of cytotoxic cells can be generated readily from T cells that have superior in vivo antitumor activity in SCID mice, as compared with LAK cells.
Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production.
Authors: Nastala C L , Edington H D , McKinney T G , Tahara H , Nalesnik M A , Brunda M J , Gately M K , Wolf S F , Schreiber R D , Storkus W J
Published: December 2022

Recent evidence supports the critical and proximate role of IL-12 in regulating both T and NK cell function during inflammation. In these studies, we evaluated the in vivo antitumor activity of murine IL-12 in murine adenocarcinoma and sarcoma models using both systemic and peritumoral administration. Antitumor effects were consistently demonstrated both in models of microdisease, in which IL-12 treatment was initiated soon after tumor inoculation (1 to 5 days), and in animals bearing large established tumors (7 to 14 days). Treatment with IL-12 markedly prolonged survival and, in most cases, caused complete tumor regression. Significant reduction in pulmonary metastases after systemic treatment was observed when treatment was delayed for 10 days after tumor inoculation. Increases in serum IFN-gamma, TNF-alpha, and nitrogen oxides were demonstrated, exceeding those observed with IL-2 treatment. Systemic administration of anti-IFN-gamma Abs before IL-12 treatment nearly completely abrogated the antitumor effect in experiments using subcutaneous tumors or pulmonary metastases. Depletion of the individual T cell subsets CD4 and CD8 by systemic administration of mAbs diminished the effectiveness of IL-12 when administered in combination. An infiltrate composed primarily of CD8+ + cells was demonstrated by using immunohistochemical analysis of tumors after IL-12 treatment. Minimal apparent toxicity was demonstrated at effective doses (0.1 to 1.0 microgram/day) of IL-12. These results indicate that IL-12 is an effective and minimally toxic antitumor agent in murine tumor models and leads to an immune-mediated rejection involving, at least in part, IFN-gamma, CD4+, and CD8+ cells. Human clinical trials of IL-12 for the treatment of malignancy are supported by these studies.
Identification of two novel regions of human IL-6 responsible for receptor binding and signal transduction.
Authors: Ehlers M , Grötzinger J , deHon F D , Müllberg J , Brakenhoff J P , Liu J , Wollmer A , Rose-John S
Published: December 2022

The pleiotropic cytokine IL-6 has been predicted to be a protein with four antiparallel alpha-helices. Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of a set of chimeric human/murine IL-6 proteins has recently allowed us to define a new region (residues Lys41-Glu95) within the IL-6 molecule as being important for receptor binding and biologic activity. We subdivided and analyzed this region, which primarily corresponds to the loop between the first and second alpha-helix of IL-6 with respect to its role in the interaction with the ligand binding subunit of the IL-6 receptor complex and with the IL-6 signal-transducing protein gp130. By construction and analysis of human/murine chimeric IL-6 molecules with only 7 to 10 amino acid residues different from human IL-6 we show that two distinct parts of this region are responsible for receptor binding and signal transduction. On the basis of the recently published structure of granulocyte-CSF, we present a three-dimensional model for the tertiary structure of IL-6, which, together with the IL-6 receptor interaction data, allows for the rational design of human IL-6 receptor antagonists.
Multiple glycosylphosphatidylinositol-anchored Ly-6 molecules and transmembrane Ly-6E mediate inhibition of IL-2 production.
Authors: Fleming T J , Malek T R
Published: December 2022

Cross-linking of Ly-6 molecules on T lymphocytes leads to IL-2 production, whereas costimulation of T cells via Ly-6A/E and the TCR inhibits IL-2 secretion. This study was initiated to determine whether there are unique structural requirements at the level of the Ly-6 molecule for its capacity to activate or block IL-2 production. Functional studies in which transfected EL-4J cells that expressed various Ly-6 proteins or chimeric Ly-6 molecules were used have demonstrated that direct activation of IL-2 secretion or inhibition of anti-CD3-induced IL-2 production occurred after mAb binding to Ly-6A/E, Ly-6C, and Ly-6G and was independent of whether the mAbs bound to amino- or carboxyl-terminal epitopes of Ly-6. The blockade of IL-2 production by costimulation with anti-CD3 and anti-Ly-6 was detected even when the addition of either mAb was temporally delayed for up to 17 h in a reciprocal fashion. Stimulation of mouse Ly-6 proteins expressed in Jurkat cells antagonized PMA/OKT3-induced IL-2 production, thus revealing that the Ly-6 inhibitory pathway is operative in human cells. EL-4J cells were also transfected with a chimeric construct in which the glycosylphosphatidylinositol anchor of Ly-6E was replaced by the transmembrane and a portion of the intracytoplasmic tail of H-2Db. Anti-Ly-6A/E also blocked anti-CD3-induced IL-2 production for these cells, although anti-Ly-6A/E failed to directly induce IL-2 secretion. Thus, anti-Ly-6A/E blockade of IL-2 production is independent of the glycosylphosphatidylinositol anchor of Ly-6E. This finding suggests that there may be aspects of signaling via Ly-6 that are solely dependent on the extracellular amino acid sequence of this protein.
TNF receptors TR60 and TR80 can mediate apoptosis via induction of distinct signal pathways.
Authors: Grell M , Zimmermann G , Hülser D , Pfizenmaier K , Scheurich P
Published: December 2022

TNF membrane receptors are usually co-expressed in many tissues but their relative contribution to cellular TNF responses is for most situations unknown. In a TNF cytotoxicity model of KYM-1, a human rhabdomyosarcoma cell line, we recently demonstrated that each of the two TNFRs is on its own capable of inducing cell death. Here we show that both receptors are able to induce apoptosis, as revealed from a similar onset of DNA fragmentation and typical morphologic criteria. To obtain additional information about the signaling pathways involved in TR60- and TR80-induced programmed cell death, we have used a series of selective inhibitors of intracellular signaling molecules. The overall pattern emerging from these experiments provides strong evidence for distinct signal pathway usage of TR60 and TR80, indicating protein kinase(s)-mediated control of TR60 signaling and a tight linkage of TR80 to arachidonate metabolism. The subsequent establishment of KYM-1-derived cell lines that display TNFR selective resistance further supports a segregation of TR60 and TR80 signaling pathways for induction of apoptotic cell death. Moreover, these results demonstrate an independent control of the distinct signaling cascades used by TR60 and TR80. This allows a highly flexible regulation of a cellular TNF response in those cases in which both receptors contribute to overall TNF responsiveness.
Differential up-regulation of the B7-1 and B7-2 costimulatory molecules after Ig receptor engagement by antigen.
Authors: Lenschow D J , Sperling A I , Cooke M P , Freeman G , Rhee L , Decker D C , Gray G , Nadler L M , Goodnow C C , Bluestone J A
Published: December 2022

Ag-pulsed B cells are potent APCs, in part, because of the ability of the Ig receptor to mediate rapid and specific Ag uptake. However, it is also known that full T cell activation requires signals delivered by costimulatory molecules, which naive B cells seem to lack. This study examines the effect Ig receptor engagement has on the expression and function of a new CD28 counter-receptor, B7-2. Unlike B7-1 (B7), B7-2 was rapidly induced on the cell surface of B cells after engagement of the Ig receptor by either anti-Ig mAbs or hen egg lysozyme (HEL) on normal and HEL-specific B cell receptor transgenic B cells, respectively. Furthermore, B7-2 expression was up-regulated on tolerant B cells isolated from HEL/anti-HEL double transgenic mice after Ag stimulation, although at lower levels than on nontolerant transgenic B cells. No significant cell surface levels of B7-1(B7) were observed under these conditions. Finally, the B7-2 molecules induced by Ig cross-linking costimulated T cell proliferation in a CD28-dependent manner, independent of B7-1(B7) expression. Thus, the effectiveness of Ag-specific B cells as APCs depends on both their enhanced Ag uptake, mediated by the B cell receptor, and immediate up-regulation of a potent costimulatory molecule, B7-2.
Expression of type 3 complement receptor on activated CD8+ T cells facilitates homing to inflammatory sites.
Authors: Nielsen H V , Christensen J P , Andersson E C , Marker O , Thomsen A R
Published: December 2022

It has been reported that Ag-primed T cells may express the type 3 complement receptor (CR3; CD11b/CD18, Mac-1), but no clear function has been ascribed to this molecule on T cells. In our study, we have used a nondepleting mAb to CR3 (5C6) to study the relevance of CR3 expression for T-cell extravasation at inflammatory foci. The in vivo administration of 5C6 significantly reduced the severity of the T cell-mediated meningitis induced by intracerebral inoculation of lymphocytic choriomeningitis virus. FACS analysis of the inflammatory exudate showed that this consists of monocytes/macrophages and CD8+ cells, the majority of which are CD11b+. Adoptive transfer experiments involving i.v. transplantation of Ag-primed donor cells revealed that preincubation of the cells with 5C6 delayed the virus-specific delayed-type hypersensitivity reaction under conditions in which the recipient delivered the nonspecific component. In contrast, preincubation with 5C6 did not inhibit the response when donor cells were injected locally. The reaction induced by locally injected, adherent cell-depleted donor cells was, on the other hand, delayed by i.v. injection of 5C6 to the recipient. These results indicate that anti-CR3 treatment inhibits extravasation of both the Ag-specific and the nonspecific cellular components of the lymphocytic choriomeningitis virus-induced, CD8+ cell-dependent inflammatory reaction. Thus, expression of CR3 seems to facilitate T cell homing to sites of inflammation.
Endogenous IL-1 is required for neutrophil recruitment and macrophage activation during murine listeriosis.
Authors: Rogers H W , Tripp C S , Schreiber R D , Unanue E R
Published: December 2022

By using a mixture of neutralizing mAbs to IL-1 alpha, IL-1 beta, and to the type I IL-1R, we previously documented a regulatory role for IL-1 in the development of anti-Listeria responses in mice. Both normal C.B-17 and severe combined immunodeficiency (SCID) mice injected with this mixture of Abs exhibit decreased resistance to Listeria. In this study, we demonstrate that the neutralization of IL-1 activity in SCID mice results in a major defect in neutrophil migration to the peritoneum, in response to Listeria infection. Moreover, anti-IL-1 treatment also inhibits Listeria-induced peripheral blood leukocytosis at all time points examined. We also show that mice injected with anti-IL-1 Abs failed to elaborate class II MHC-positive peritoneal macrophages in vivo at any time during Listeria infection. Even though peritoneal macrophages from anti-IL-1-treated Listeria-infected mice are not activated to express MHC class II molecules, IFN-gamma production in vivo is normal. Moreover, the macrophages are unresponsive to IFN-gamma in vitro, as assayed by MHC class II expression, even when rIL-1 is added. rIL-1 also is unable to increase the expression of IFN-gamma-induced surface class II MHC molecules on resident peritoneal macrophages in vitro. These results indicate that endogenously produced IL-1 plays an important role in Listeria-dependent neutrophil migration, increase in blood leukocyte number, generation of MHC class II-positive macrophages in vivo, and macrophage responsiveness to IFN-gamma.
Expression of MUC1 on myeloma cells and induction of HLA-unrestricted CTL against MUC1 from a multiple myeloma patient.
Authors: Takahashi T , Makiguchi Y , Hinoda Y , Kakiuchi H , Nakagawa N , Imai K , Yachi A
Published: December 2022

Polymorphic epithelial mucin (MUC1) was detected in myeloma cells and in sera of multiple myeloma patients. HLA-unrestricted CTL that recognize tumor-associated epitopes on MUC1 has been shown to be induced from breast and pancreas cancer patients. To investigate whether such CTL can also be induced from multiple myeloma patients, an allogeneic mixed leukocyte tumor cell culture was performed. PBMCs of a multiple myeloma patient were stimulated by different allogeneic breast carcinoma and myeloma cell lines. The cultured PBMCs were proliferated and a CTL line TN was established. TN exclusively expressed TCR-alpha/beta, CD3, and CD8. TN lysed breast carcinoma and myeloma cell lines but did not lyse K562, which is sensitive to NK cells. The cytotoxicity of TN was inhibited by anti-CD3 Abs but not by anti-HLA Abs. Thus, the TCR-alpha/beta was considered to be involved in the recognition of the target cells but HLA was not. Furthermore, TN lysed transformed mouse fibroblast cells transfected with MUC1 cDNA, suggesting that this CTL line recognizes MUC1 directly. Thus, it is concluded that precursors of HLA-unrestricted and anti-MUC1 reactive CTL could exist in the peripheral blood of multiple myeloma patients and that myeloma cells can express epitopes on MUC1, which can be recognized by the CTL.
Resistance of Actinobacillus pleuropneumoniae to bactericidal antibody and complement is mediated by capsular polysaccharide and blocking antibody specific for lipopolysaccharide.
Authors: Ward C K , Inzana T J
Published: December 2022

Actinobacillus pleuropneumoniae is resistant to complement-mediated killing, even in the presence of specific Ab. Our studies focused on identifying the mechanism(s) responsible for this resistance. Encapsulated A. pleuropneumoniae was susceptible to killing in precolostral calf serum (PCS) but not in normal serum as a complement source in the presence of anti-capsular polysaccharide (CP) IgG. In contrast, two capsule-deficient mutants were sensitive to killing in normal serum and one was sensitive to killing in PCS alone. Electron microscopy demonstrated that A. pleuropneumoniae serotype 5a synthesized a thick, adherent CP that bound anti-CP Ab distant from the outer membrane. The CP of A. pleuropneumoniae did not prevent complement activation or the attachment of C3 to the cell surface. However, the CP did limit the amount of C9, a component of the membrane attack complex, that bound to A. pleuropneumoniae in PCS. A second mechanism of serum resistance was a result of an LPS-specific Ab present in the IgG fractions of normal swine serum, swine anti-K17 serum, and guinea pig anti-K17 LPS that blocked anti-CP IgG complement-mediated killing of A. pleuropneumoniae. Incubation of swine anti-K17 IgG with purified K17 LPS depleted Abs specific for K17 LPS but not for K17 proteins and removed all blocking activity. Immune swine serum containing this blocking Ab reduced the deposition of C9 on A. pleuropneumoniae in the presence of anti-CP IgG and also directed the deposition of C9 to sites on the bacteria in which the bound C9 was easily eluted. Thus, CP and anti-LPS Ab may act synergistically or at different stages of infection to limit the ability of complement to eliminate A. pleuropneumoniae.
Comparison of human eosinophil and neutrophil adhesion to endothelial cells under nonstatic conditions. Role of L-selectin.
Authors: Knol E F , Tackey F , Tedder T F , Klunk D A , Bickel C A , Sterbinsky S A , Bochner B S
Published: December 2022

To simulate adhesion that occurs under conditions of flow, we investigated the attachment of eosinophils to endothelium under rotational conditions. Tissue-culture plates containing monolayers of HUVEC were placed on a horizontal rotator (80 revolutions per minute (rpm)), and equal numbers of purified human eosinophils or neutrophils were added to separate wells at 4 degrees C. Binding of eosinophils and neutrophils to unstimulated endothelial cells was 15 +/- 3 and 31 +/- 11 cells/four high power fields (HPF), respectively. After preincubation of HUVEC with IL-1 beta (1 ng/ml, 4 h, 37 degrees C), adhesion increased to 56 +/- 4 and 290 +/- 26 cells/four HPF, respectively (p < 0.0002 for both, n = 8-14). Eosinophils with reduced levels of L-selectin (blood eosinophils activated in vitro or eosinophils obtained from bronchoalveolar lavage (BAL) performed after segmental lung allergen challenge of allergic subjects) demonstrated reduced binding under rotating conditions. Several L-selectin Abs inhibited adhesion of eosinophils and neutrophils (e.g., LAM1-3: 43 +/- 14% vs 63 +/- 3% inhibition; LAM1-6: 73 +/- 5% vs 36 +/- 6% inhibition, respectively, n > or = 6). Interestingly, one additional L-selectin Ab, LAM1-11, inhibited eosinophil but not neutrophil adhesion (51 +/- 2% vs 1 +/- 7% inhibition, respectively, n > or = 5). We conclude that eosinophils, like neutrophils, use L-selectin to bind to activated endothelial cells under conditions of flow, although mAb LAM1-11 can selectively inhibit eosinophil attachment to stimulated endothelial cells in vitro, suggesting different functional epitopes on L-selectin among eosinophils and neutrophils.
TNF and its receptor antibody agonist differ in mediation of cellular responses.
Authors: Totpal K , LaPushin R , Kohno T , Darnay B G , Aggarwal B B
Published: December 2022

TNF binds to two distinct receptors designated p60 and p80. Because Abs to the p60 receptor (anti-p60) can mimic TNF, we therefore compared the cellular signaling of TNF with that of anti-p60. We demonstrate both qualitative and quantitative differences between TNF and anti-p60. HepG2 cells, which express the p60 receptor, were found to be completely resistant to TNF but highly sensitive to the antiproliferative effects of anti-p60. In contrast, normal fibroblasts were found to be several fold more sensitive to TNF than to anti-p60. Several other epithelial cell lines that also express primarily the p60 receptor showed quantitative differences in mediation of cellular responses by TNF and anti-p60. The blocking of the p60 receptor by TNF had no effect on the response of HepG2 cells to anti-p60, suggesting a difference in their binding sites. Anti-p60, however, inhibited the effect of TNF on fibroblasts. Ab against the p80 receptor had no effect by itself or on the effect of TNF and anti-p60. The difference in the response to TNF and anti-p60 could not be correlated to the differences in the level of expression of p60 receptor on these cells. Furthermore, cycloheximide potentiated the TNF-mediated effect but not that mediated through anti-p60, thus also indicating a difference in the mechanism of action of these two agents. Overall, these results demonstrate that TNF and anti-p60, although both working through the p60 receptor, differ in their cellular signaling.
Autoantibodies to human nuclear lamin B2 protein. Epitope specificity in different autoimmune diseases.
Authors: Brito J , Biamonti G , Caporali R , Montecucco C
Published: December 2022

The nuclear lamina of mammalian cells consists of three major proteins, lamins A, C, and B, and a fourth minor protein, lamin B2. Lamins belong to the family of intermediate filaments and are highly similar both in structure and primary sequence. They are organized in three well-defined domains: 1) a central alpha-helical rod, which is a secondary structure shared by all types of intermediate filaments, formed by three alpha-helices (coils 1A, 1B, and 2) and surrounded by 2) an amino-terminal head and 3) a carboxyl-terminal tail. Autoantibodies toward major lamin have been described previously in sera from patients with different autoimmune diseases. We chose an epitope mapping approach to further characterize the autoimmune response to nuclear lamin. Different lamin B2 domains were expressed as fusion proteins with the glutathione S-transferase and then used in immunoblotting experiments to analyze sera from patients with autoimmune diseases (chronic active hepatitis, SLE, rheumatoid arthritis, and polymyalgia rheumatica) and from healthy subjects. At a 1:1000 dilution, none of the control sera recognized any of the recombinant polypeptides. Conversely, reactive sera were present in all groups of patients. The ability to recognize a protein domain seemed to differ with the pathology. Most chronic active hepatitis sera were reactive to two or more lamin domains and reacting SLE sera always gave positive signals to coil 2 and/or coil 1B. Coil 2 was preferentially recognized by rheumatoid arthritis sera. Polymyalgia rheumatica sera differed from all of the others because of their low reactivity to the rod domain and preference for the C terminus, a lamin-specific domain.
Prevention of mercuric chloride-induced nephritis in the brown Norway rat by treatment with antibodies against the alpha 4 integrin.
Authors: Molina A , Sánchez-Madrid F , Bricio T , Martín A , Barat A , Alvarez V , Mampaso F
Published: December 2022

HgCl2 induces the synthesis of anti-GBM Abs with the development of glomerular and interstitial nephritis, as well as proteinuria, in the Brown Norway rat. The development of this autoimmune disease is a consequence of the appearance of an autoreactive T cell subset-inducing activation of B cells. The administration to mercury-treated rats of the mouse anti-human VLA alpha 4 HP2/1 mAb, which cross-reacts with the rat homologue integrin, completely abrogated the interstitial cell infiltrates. As demonstrated by peripheral blood analysis, this effect is not a result of the depletion of circulating leukocytes or leukocyte subsets. Interestingly, the administration of Abs specific for the alpha 4 integrin also highly reduced anti-GBM Ab synthesis, thus preventing detectable glomerular deposits and proteinuria. Our results confirm that in vivo alpha 4 functions in adhesive interaction of circulating leukocytes and vascular endothelium, and is centrally important in the extravasation and migration of T lymphocytes to sites of tissue injury. We also found a complete absence of interstitial cell infiltrates, together with a positive glomerular IgG lineal deposition pattern, when anti-GBM Abs were passively transferred to rats pretreated with anti-alpha 4 mAb, thus indicating an independent role of alpha 4 integrin in both extravasation of immune cells and production of autoantibodies. Furthermore, these in vivo findings provide preliminary evidence for the participation of the VLA-4 integrin in mediating the intercellular interaction of leukocytes regulating the production of Abs, most likely through the existence of additional yet unknown ligand(s).
Resistance of CTL to perforin-mediated lysis. Evidence for a lymphocyte membrane protein interacting with perforin.
Authors: Müller C , Tschopp J
Published: December 2022

Cytotoxic T lymphocytes are highly resistant to killing by their own cytolytic protein perforin. We have investigated the molecular basis of this self-protection mechanism. CTL withstood high dosages of perforin, when complete lysis was obtained with various tumor target cell lines. A peptide-specific CTL clone readily lysed tumor targets presenting the peptide, but was unable to kill the peptide-presenting CTL in spite of equal degranulation. With streptolysin O, a pore-forming lytic protein of bacterial origin, no difference in susceptibility between the various targets was detected. Perforin was radioactively labeled by using the Bolton and Hunter method without any loss of its lytic activity. Experiments performed at 4 degrees C revealed that, on all of the cells studied, binding of the labeled perforin to the membrane is reversible and strictly Ca(2+)-dependent. After a short exposure to 37 degrees C, perforin was no longer dissociated from cell membranes by EDTA. Although no correlation between susceptibility and the extent of perforin binding was detected, differences in the conformation of membrane-bound perforin were observed. Perforin adsorbed to resistant cells was cleaved by trypsin into a 55-kDa (C-terminal) and a 10- to 15-kDa (N-terminal) fragment, whereas this cleavage was not found on tumor cell-bound perforin. A portion of perforin was included in the Triton X-114 detergent phase at 4 degrees C on resistant CTLs only. Our results are compatible with the notion that a protective molecule, specifically expressed on CTLs, interacts with perforin, thereby rendering it lytically inactive.
Induction of IL-8 expression in T cells uses the CD28 costimulatory pathway.
Authors: Wechsler A S , Gordon M C , Dendorfer U , LeClair K P
Published: December 2022

IL-8, a potent chemotactic factor for neutrophil granulocytes and lymphocytes, is a proinflammatory cytokine secreted by a variety of cell types, including T cells. Stimulation of the CD28 cell surface molecule delivers costimulatory signals essential for lymphokine production in activated T cells via a conserved sequence element found in the promoter of several lymphokine genes. Anti-CD28-stimulated T cells produced significant amounts of IL-8; additionally, costimulation with anti-CD3 and anti-CD28 Abs resulted in a synergistic induction of IL-8 secretion. Sequence homology, single nucleotide mutations, and anti-CD28 Ab stimulation studies established that the NF-kappa B-like sequence in the promoter of the IL-8 gene functioned as a CD28 response element. Furthermore, cyclosporin A, but not rapamycin, blocked the synergistic induction of IL-8 expression achieved with anti-CD3 and anti-CD28 costimulation. The involvement of a CD28 response element in the induction of IL-8 expression in activated T cells may provide new insights into the pathogenesis and persistence of immune disorders characterized by increased levels of IL-8, such as psoriasis and rheumatoid arthritis.
Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii.
Authors: Gazzinelli R T , Wysocka M , Hayashi S , Denkers E Y , Hieny S , Caspar P , Trinchieri G , Sher A
Published: December 2022

In vitro and in vivo studies were performed to assess the involvement of IL-12 in resistance to acute and chronic infection with an avirulent strain of Toxoplasma gondii. Our previous findings implicated macrophages as a major source of parasite-induced IL-12. This finding was confirmed by showing that peritoneal macrophages exposed to either live parasites or soluble tachyzoite Ags produce IL-12 protein. In mice, increased expression of IL-12 (p40) mRNA in both spleen and peritoneal cells was detected as early as 2 days postinfection. Treatment with neutralizing mAbs against IL-12 increased the susceptibility of C57BL/6, BALB/c, and severe combined immunodeficient (SCID) mice to acute infection, which resulted in 100% mortality within the first 15 days after parasite inoculation. In contrast, neutralization of endogenously produced IL-12 had no effect when given during chronic infection. In agreement with the survival data, treatment with anti-IL-12 resulted in decreased IFN-gamma and enhanced Th2 (IL-4 and IL-10) cytokine synthesis by splenocytes when given during acute, but not chronic, toxoplasmosis. Sorting experiments on spleen cells from acutely infected mice indicated that both CD4+ lymphocytes and NK1.1+/CD3- cells contribute to the early IFN-gamma response. In contrast, CD4+ cells were found to be the major source of the cytokine during chronic disease. Together, these results suggest that the stimulation of macrophage-derived IL-12 plays a major role in both the induction of resistance and Th1 cell subset selection in acute T. gondii infection, but may not be required to maintain established immunity.
Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages.
Authors: Xu S , Cooper A , Sturgill-Koszycki S , van Heyningen T , Chatterjee D , Orme I , Allen P , Russell D G
Published: December 2022

Despite the potential role of the macrophage in the eradication of invading microbes, Mycobacterium species have evolved mechanisms to ensure their survival and replication inside the macrophage. Particles phagocytosed by macrophages normally will be delivered into acid lysosomal compartments for degradation. Mycobacterium must, in some way, avoid this fate by modulation of their phagosome. Immunoelectron microscopy of macrophages infected with Mycobacterium avium or Mycobacterium tuberculosis indicates that the vacuolar membrane surrounding the bacilli possesses the late endosomal/lysosomal marker, LAMP-1 (lysosomal-associated membrane protein-1), but lacks the vesicular proton-ATPase. Analysis of the intersection of the bacteria-containing vacuoles with the endocytic network of the macrophage supports previous studies indicating that these bacilli restrict the fusion capability of their intracellular compartments. The occurrence of vesicles containing lipoarabinomannan, discrete from those containing Mycobacterium, indicate that material does traffic out from the mycobacterial vacuole. To compensate for this loss of membrane, the vacuole must remain dynamic and fuse with LAMP-1-containing vesicles to maintain the density of this marker.
Molecular cloning and identification of the porcine cytolytic trigger molecule G7 as a Fc gamma RIII alpha (CD16) homologue.
Authors: Halloran P J , Sweeney S E , Strohmeier C M , Kim Y B
Published: December 2022

The G7 mAb binds a previously unidentified cytolytic trigger molecule on porcine NK cells and polymorphonuclear cells (PMN). This mAb has been shown to almost completely block PBL-mediated Ab-dependent cellular cytotoxicity and inhibit PMN-mediated Ab-dependent cellular cytotoxicity by approximately one-half, indicating a close functional association of the G7 molecule (to which the G7 mAb binds) with Fc gamma R function. This study was designed to identify the G7 molecule through molecular cloning of its cDNAs. The G7 protein was purified and peptide fragments subjected to amino acid sequencing. This information was then used in the PCR amplification of three cDNA fragments encoding a full-length G7 polypeptide. Amino acid sequences corresponding to peptides derived from G7 are observed within the deduced amino acid sequence of the individual clones confirming the specificity of G7 purification and PCRs. The specificity is also confirmed by reactivity of an antiserum raised against a synthetic G7 peptide to immunoprecipitated G7. Northern blot analysis indicates that a 0.9-kb G7 mRNA is expressed in porcine PMN, PBMC, macrophage, spleen, and lymph nodes but not in thymus or liver. Nucleotide and deduced amino acid sequence analyses indicate that G7 represents a porcine homologue to human Fc gamma RIIIA alpha. This is especially important in that G7 has been previously demonstrated to exist in a molecular complex with a novel molecule, PNK-E. The data suggests the presence of a novel Fc gamma RIII alpha molecular complex (G7/PNK-E) in the porcine system.