bluuebunny/crossref_metadata_2025 huggingface dataset:
Transforming growth factor-beta and IL-1 beta act in synergy to enhance IL-6 secretion by the intestinal epithelial cell line, IEC-6.
Authors: McGee D W , Beagley K W , Aicher W K , McGhee J R
Published: January 2023

Intestinal epithelial cells are a potentially important source for a number of cytokines that may modulate the immune response at the intestinal mucosa. We have recently begun to study the mechanisms that regulate IL-6 production by intestinal epithelial cells using the nontransformed crypt-like rat intestinal epithelial cell line IEC-6 as a model. Culture of the IEC-6 cells with human rIL-1 beta resulted in an enhanced secretion of IL-6 by the cells. RT-PCR analysis of IL-1 beta-treated cells showed an enhanced level of IL-6 mRNA at 4 h, suggesting that IL-1 beta enhanced IL-6 gene expression. In a previous study, transforming growth factor-beta (TGF-beta 1) was also found to enhance IL-6 secretion by the IEC-6 cells and because both IL-1 beta and TGF-beta may be present in inflamed mucosal tissue, the effect of adding both cytokines together was next investigated. Culture of the IEC-6 cells with both TGF-beta 1 and IL-1 beta resulted in a synergistic enhancement of IL-6 secretion that was seen even at high levels of both TGF-beta and IL-1 beta. IL-6 mRNA levels from cells treated with both TGF-beta 1 and IL-1 beta were also determined to be enhanced when compared to that of cells treated with IL-1 beta or TGF-beta 1 only, as determined by RT-PCR analysis. Pretreatment of the IEC-6 cells with TGF-beta 1 for 2 or 3 days before addition of the IL-1 beta induced the IEC-6 cells to differentiate and become more sensitive to stimulation by IL-1 beta. Subsequent experiments determined that TGF-beta enhanced the capacity of the IEC-6 cells to bind labeled IL-1 beta indicating that TGF-beta may have enhanced the expression of IL-1 receptors on the cells. These results suggest that the intestinal epithelial cell may represent an important source of IL-6 in inflammatory responses at the intestinal mucosa and that TGF-beta could potentiate this function.
Production of minor lymphocyte stimulatory-1a antigen from activated CD4+ or CD8+ T cells.
Authors: Arase-Fukushi N , Arase H , Ogasawara K , Good R A , Onoé K
Published: January 2023

Minor lymphocyte stimulatory (Mls) Ag are super Ag that stimulate a high proportion of T cells of a specific TCR V beta family. One of the super Ag, Mls-1a, which is recognized mainly by TCR V beta 6+ and V beta 8.1+ T cells, has recently been linked to the response to product of the open reading frame in 3'-long terminal repeat of endogenous mammary tumor virus, MTV-7. It is quite certain that B cells are able to produce and also to present the Mls-1a Ag. However, it remains to be determined whether other cell types, especially T cells, produce Mls-1a Ag. In this study using highly purified T cell subpopulations, capacity to produce Mls-1a Ag was analyzed by calculating the proportion of Mls-1a reactive V beta 6+ or V beta 8.1+ T cells in responding cell populations. We found that nonstimulated CD8+ T cells produced a low amount of Mls-1a Ag, and the capacity to do so was considerably increased by stimulation with immobilized anti-TCR mAb. By contrast, nonstimulated CD4+ T cells did not produce Mls-1a Ag at all. Even when CD4+ T cells were activated via TCR signaling with immobilized anti-TCR mAb, CD4+ T cells did not produce Mls-1a Ag. However, CD4+ T cells primed with conventional Ag in vivo produced Mls-1a Ag on restimulation with that specific Ag in vitro. These findings indicate that not only CD8+ T cells but also CD4+ T cells can produce Mls-1a Ag on appropriate stimulation, although different mechanisms for Mls-1a production may operate between the CD4+ and CD8+ T cells.
The expression of CD4 by T cell precursors resident in both the thymus and the bone marrow.
Authors: Chervenak R , Dempsey D , Soloff R , Wolcott R M , Jennings S R
Published: January 2023

A very small fraction of thymocytes has recently been identified that expresses low levels of CD4 in the absence of CD8, CD3, or a TCR. These CD4lo thymocytes appear to be the precursors of the early CD4-CD8-CD3- thymic subset and contain most of the T cell progenitor activity found within the thymus. Here, we examined adult bone marrow for the presence of a similar population of cells and found that 0.5% to 3.5% of C58/J bone marrow cells express low, but detectable levels of CD4 (CD4lo) at the cell surface in the absence of CD3. These CD4lo bone marrow cells display pre-T cell activity, in that they are able to repopulate the thymus of irradiated recipient mice after intrathymic transfer. Moreover, we found that most of pre-T cell activity found in the bone marrow is contained within the CD4lo expressing subset of marrow cells. Although the CD4lo cells found in both the thymus and bone marrow display pre-T cell activity, the CD4lo cells from these two sites showed pronounced differences with respect to their ability to respond to specific cytokine stimulation in vitro. Bone marrow-resident CD4lo cells proliferated in response to both IL-3 and mast cell growth factor in vitro, whereas CD4lo cells isolated from the thymus did not. Furthermore, CD4lo bone marrow cells, grown in media containing IL-3 and mast cell growth factor, retained their pre-T cell activity, indicating that CD4lo cells with pre-T cell capabilities were among the IL-3 and mast cell growth factor-responsive cells. These data suggest that although pre-T cells in bone marrow share the CD4lo phenotype with their intrathymic counterparts, they may be fundamentally different with respect to the environmental factors that control their growth.
TGF-beta is a bidirectional modulator of cytokine receptor expression on murine bone marrow cells. Differential effects of TGF-beta 1 and TGF-beta 3.
Authors: Jacobsen S E , Ruscetti F W , Roberts A B , Keller J R
Published: January 2023

Transforming growth factor beta (TGF-beta), an immunomodulator, has inhibitory as well as stimulatory effects on bone marrow cells. In this study, we demonstrate that TGF-beta 1 also is a bidirectional modulator of CSF receptor expression on murine bone marrow cells. TGF-beta 1 up-regulated granulocyte-macrophage (GM)-CSF receptor expression in a time- and dose-dependent manner, with a maximum up-regulation of 64% by 48 h at 20 ng/ml. In contrast, TGF-beta 1 down-modulated IL-3 and CSF-1 receptor expression by 54 and 55%, respectively, by 24 h. TGF-beta 1 did not affect G-CSF receptor expression, in agreement with its inability to affect G-CSF-induced proliferation. The CSF receptor modulation induced by TGF-beta 1 preceded its effects on CSF-stimulated proliferation. The effects of TGF-beta on CSF receptor expression were isoform dependent, thus TGF-beta 3 was a 10-fold more potent inhibitor of both IL-3-induced colony formation and IL-3 receptor expression than TGF-beta 1, whereas TGF-beta 1 was a more potent stimulator of GM-CSF-stimulated colonies and GM-CSF receptor expression than TGF-beta 3. Therefore, the ability of TGF-beta to modulate the CSF receptor density/cell and/or the actual number of progenitors expressing CSF receptors directly correlates with the multifunctional effects of TGF-beta in hematopoiesis.
Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor.
Authors: Tartaglia L A , Goeddel D V , Reynolds C , Figari I S , Weber R F , Fendly B M , Palladino M A
Published: January 2023

TNF-alpha can enhance the proliferation of human thymocytes stimulated by the comitogen Con A. To determine which of the two different TNF receptors is responsible for signaling this cellular response, we investigated the proliferation of human thymocytes in response to agonistic antibodies specific for the two TNF receptor types. In contrast to previously examined TNF activities in human cells, thymocyte proliferation was stimulated in response to rabbit polyclonal antibodies directed against the 75-kDa TNF receptor (TNF-R2), but not those directed against the 55-kDa TNF receptor (TNF-R1). Lymphotoxin (TNF-beta) was also shown to stimulate human thymocyte proliferation, demonstrating that TNF-beta can initiate a biologic response that is mediated by TNF-R2. TNF-R2-mediated T-cell proliferation was not restricted to the immature T cells within the thymus, as the anti-TNF-R2 antibodies also stimulated the proliferation of peripheral T cells. As a first step toward identifying a specific agonist of TNF-R2 with therapeutic potential, 10 anti-TNF-R2 mAb were examined for potential agonist activity. Nine of these significantly stimulated human thymocyte proliferation with maximal responses ranging from twofold to significantly greater than that obtained with TNF-alpha by itself.
Construction of a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments.
Authors: Akamatsu Y , Cole M S , Tso J Y , Tsurushita N
Published: January 2023

A naive combinatorial Ig library was constructed from semi-synthetic V genes consisting of human genomic V segments and synthetic CDR3 fragments. VH and V kappa segments were amplified from human genomic DNA by polymerase chain reaction using V subgroup-specific primers. The amplified VH and V kappa segments were combined with synthetic oligonucleotides containing a J region and CDR3 with amino acid sequence variations, resulting in complete V genes. These V genes were cloned into a phagemid expression vector in a single-chain form fused to the carboxyl-terminus of the M13 minor coat protein III. Phagemid particles displaying the single chain hybrid proteins on their surface were screened with Con A as Ag. Several clones showing specific binding to Con A were obtained after four rounds of selection and were further analyzed for their binding properties and DNA sequences. This method provides a novel way to create a naive combinatorial library without using mRNA from B lymphocytes as template. The method should be useful to isolate human antibodies that react with self-Ag.
Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells.
Authors: Morton H C , Atkin J D , Owens R J , Woof J M
Published: January 2023

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.
Killing of Staphylococcus aureus by tumor necrosis factor-alpha-activated neutrophils. The role of serum opsonins, integrin receptors, respiratory burst, and degranulation.
Authors: Ferrante A , Martin A J , Bates E J , Goh D H , Harvey D P , Parsons D , Rathjen D A , Russ G , Dayer J M
Published: January 2023

We have examined the effects of TNF priming on the killing of Staphylococcus aureus by human neutrophils. In the absence of serum opsonins, neutrophils failed to kill S. aureus, and TNF priming did not induce the cells to become bactericidal. Normal human serum, containing complement activity, promoted the killing of the bacteria by neutrophils. Pretreatment of neutrophils for 30 min with TNF significantly enhanced their bactericidal activity. The effects of TNF on neutrophil bactericidal activity was dependent on serum concentration and the degree of enhancement induced increased up to a concentration of 1%. The kinetics of bacterial killing showed that TNF-only enhanced the initial rate of killing, over the first 30 min. Little killing of bacteria occurred in the presence of complement-inactivated serum, and TNF did not stimulate this killing. These results suggest that TNF enhances the neutrophil complement-dependent killing of S. aureus. TNF increased the expression of CR3 (CD11b/CD18) and CR4 (P150, 95; CD11c/CD18) adhesion receptors but not LFA-1 (CD11a/CD18); and mAb against the alpha-chain of either CR3 or CR4 but not LFA-1 prevented the enhancing effects of TNF on the neutrophil bactericidal activity.
Role of kinins in the vascular extravasation evoked by antigen and mediated by tachykinins in guinea pig trachea.
Authors: Bertrand C , Nadel J A , Yamawaki I , Geppetti P
Published: January 2023

Tachykinins released from sensory nerves mediate, at least in part, the plasma extravasation induced by allergen challenge to the airways of sensitized guinea pigs. We investigated the role of kinins in this activation of sensory nerves. We found that the increase in Evans blue dye extravasation evoked by aerosol of bradykinin (100 microM, 2 min) in the presence of phosphoramidon (2.5 mg/kg, i.v.) was abolished completely by the selective B2 bradykinin antagonist, HOE 140 (0.1 mumol/kg, i.v.), and was inhibited (60%) by the selective NK1 tachykinin receptor antagonist, CP-96,345 (2 mumol/kg, i.v.). Plasma extravasation evoked by aerosolized substance P (10 microM/kg, 2 min) in presence of phosphoramidon was abolished by CP-96,345, but was not affected by HOE 140. The extravasation of the Evans blue dye evoked by OVA (5%, 2 min) in sensitized guinea pigs was reduced by HOE 140 (45%) when the animals were perfused after 5 min and by 39% when perfusion was performed at 10 min. In the presence of phosphoramidon, the response to OVA at 10 min was reduced by 57% by HOE 140 and by 72% by CP-96,345. The combination of CP-96,345 and HOE 140 did not further increase the inhibition obtained with CP-96,345 alone. The results provide evidence that the activation of sensory nerves that contribute to Ag-evoked plasma extravasation is due to kinin release. The contribution of this cascade of events may be exaggerated in pathophysiologic conditions in which neutral endopeptidase is down-regulated.
The effects of stem cell factor on the ultrastructure of Fc epsilon RI+ cells developing in IL-3-dependent murine bone marrow-derived cell cultures.
Authors: Rottem M , Goff J P , Albert J P , Metcalfe D D
Published: January 2023

Stem cell factor (SCF) is known to alter the proteoglycans, proteases, and cytokines synthesized by mast cells and to activate basophils. To determine whether SCF could also effect the ultrastructural characteristics of basophils and mast cells, we examined the ultrastructure of these Fc epsilon RI+ cells over 42 days in IL-3-dependent murine bone marrow-derived cell cultures in the presence or absence of SCF. Initial experiments revealed that the addition of SCF to IL-3-dependent cells enhanced their proliferative rate without influencing the percentage of Fc epsilon RI+ or metachromatic cells. We next isolated the Fc epsilon RI+ cells using flow cytometry. Light microscopy of these cells revealed mixed cultures of both immature and mature mast cells and basophils with mature mast cells predominating by 3 wk. One hundred to 150 Fc epsilon RI+ cells were then photographed by electron microscopy at 3, 10, 21, and in some cases, 42 days of culture, and the ultrastructure of each cells was evaluated by morphometry. Each cell was scored as a mast cell or basophil using standard criteria. Analysis of this data revealed that SCF in the presence of IL-3 promoted the development of mast cells, although a significant number of basophils were noted at day 21 but were absent by day 42. When bone marrow cells cultured in IL-3 + SCF were compared with cells cultured in IL-3 alone, a significant decrease in cell and nuclear size and granule number and size was noted in both mast cells and basophils cultured in IL-3 + SCF, and basophils and mast cells under these conditions most resemble their in vivo counterparts. Thus, SCF in the presence of IL-3 increases the ratio of mast cells to basophils and alters the ultrastructural characteristics of mast cells and basophils toward a more mature phenotype.
Study of natural lipocortin I. A potent mediator for macrophage-mediated immunosuppression in tumor-bearing mice.
Authors: Sakata T , Iwagami S , Tsuruta Y , Suzuki S , Suzuki R
Published: January 2023

We previously reported a possible role of lipocortin I secreted from Mac-1+, -2+ macrophages in immunosuppression in tumor-bearing mice. In this study, we purified natural lipocortin I from the spleens of tumor-bearing mice and compared its immunosuppressive activity with recombinant lipocortin I produced by Escherichia coli. The culture supernatants of splenic macrophages from tumor-bearing mice suppressed the mitogenic responses in splenic lymphocytes. The culture supernatants contained higher levels of lipocortin I, but not PGE2, which was considered as a major immunosuppressive factor. Anti-lipocortin I antiserum neutralized these inhibitory activities. Natural lipocortin I purified from the spleens of tumor-bearing mice showed a potent suppressive activity, which was > or = 200 times higher than that of recombinant mouse lipocortin I at protein level. Alkaline phosphatase treatment of natural lipocortin I decreased immunosuppressive activity to the level of recombinant lipocortin I. N-glycosidase F treatment failed to decrease the immunosuppressive activities. These results suggest that the protein modification of lipocortin I, possibly phosphorylation but not glycosylation, is critical for the potent immunosuppressive activity in tumor-bearing mice.
Immortalization with herpesvirus saimiri modulates the cytokine secretion profile of established Th1 and Th2 human T cell clones.
Authors: De Carli M , Berthold S , Fickenscher H , Fleckenstein I M , D'Elios M M , Gao Q , Biagiotti R , Giudizi M G , Kalden J R , Fleckenstein B
Published: January 2023

T blasts of six established human CD4+ T cell clones with defined Ag specificity and cytokine secretion profile (3 Th1 and 3 Th2) were immortalized with Herpesvirus saimiri (HVS) and compared with their uninfected counterparts for their ability to proliferate, produce cytokines, and express cytolytic activity. HVS-transformed Th1 and Th2 clones neither substantially changed their original surface markers nor lose their ability to proliferate in response to their specific Ag but did acquire the ability to proliferate in response to contact signals delivered by SRBC or autologous APC alone. In addition, transformation by HVS substantially enhanced the lectin-dependent cytolytic activity of Th1 clones and enabled noncytolytic Th2 clones to exert cytolytic activity. HVS-transformed Th1 clones but not their uninfected counterparts spontaneously transcribed and secreted Th1-type cytokines (IL-2, IFN-gamma, and TNF-beta) and such a production was further enhanced by stimulation with either SRBC or PMA plus anti-CD3 mAb. HVS transformed but not uninfected Th2 clones constitutively expressed both IL-4 and IL-2 mRNA and secreted IFN-gamma. Stimulation with PMA plus anti-CD3 mAb induced uninfected Th2 clones to secrete high amounts of IL-4 and IL-5 but not Th1-type cytokines, whereas the same HVS-transformed Th2 showed minimal IL-4 and IL-5 secretion with concomitant high production of IL-2, IFN-gamma, and TNF-beta. Transformation by HVS also resulted in up-regulation of TNF-alpha and IL-3 production by both Th1 and Th2 clones. The ongoing proliferation of HVS-transformed clones was partially inhibited by either anti-IL-2 or anti-IL-3 antibodies and virtually abolished by the combined addition of the two anticytokine antibodies, suggesting that both IL-2 and IL-3 can function as autocrine growth factors for HVS-transformed Th1 and Th2 clones.
Longitudinal exposure of human T lymphocytes to weak oxidative stress suppresses transmembrane and nuclear signal transduction.
Authors: Flescher E , Ledbetter J A , Schieven G L , Vela-Roch N , Fossum D , Dang H , Ogawa N , Talal N
Published: January 2023

Products of polyamine oxidase activity, at micromolar levels and during a period of 2 to 3 days, down-regulate IL-2 mRNA levels and activity in human lymphocytes. We studied whether this suppression was associated with signal transduction abnormalities. We found that polyamine oxidase activity suppresses both anti-CD3-induced IL-2 production and protein tyrosine phosphorylation. Polyamine oxidase activity also caused a reduction in intracellular calcium mobilization after mitogenic stimulation. The most distal step of CD3-mediated signal transduction is dependent upon transcription factors that regulate a set of genes, including IL-2. We found that polyamine oxidase-treated cells exhibited very low DNA binding activity of two such factors: NFAT and NF-kappa B. On the other hand, AP-1 DNA binding activity was enhanced in polyamine oxidase-treated cells, suggesting a possible role for AP-1 in the human lymphocyte stress response. In accordance with the oxidation dependence of this suppressive mechanism, N-acetylcysteine (NAC; an antioxidant) significantly reversed the polyamine oxidase effects on lymphokine production and signal transduction. These results suggest that NAC contributes, under oxidizing conditions, to the preservation of immune function. In summary, our data suggest that chronic low-level oxidative stress, via suppression of mitogen-induced transmembrane signaling (protein-tyrosine phosphorylation and calcium mobilization), causes a decrease in the DNA binding activity of transcription factors that regulate the IL-2 gene. This results in decreased IL-2 production.
Activation of MAP2-kinase in B lymphocytes by calcium ionophores.
Authors: Franklin R A , Tordai A , Mazer B , Terada N , Lucas J J , Gelfand E W
Published: January 2023

The role of increases in intracellular calcium levels on tyrosine phosphorylation in human B lymphocytes was studied. Stimulation of normal, resting B lymphocytes or B lymphoblastoid cells with the calcium ionophores ionomycin or A23187 induced the tyrosine phosphorylation and the enzymatic activation of microtubule-associated protein-2 kinase (MAP2-K). Treatment of these cells with PMA induced tyrosine phosphorylation of a protein with the identical mobility, as well as the enzymatic activation of MAP2-K. Stimulation of these cells with ionomycin also resulted in increased ribosomal S6 kinase activity. Activation of MAP2-K in B lymphocytes by calcium ionophore was rapid (detectable within 1 min), transient (returning to background levels by 45 min), and dependent on extracellular calcium. These results demonstrate that transmembrane calcium flux induced by calcium ionophore results in the tyrosine phosphorylation and enzymatic activation of MAP2-K in human B cells.
Active induction of unresponsiveness (tolerance) to DNFB by in vivo ultraviolet-exposed epidermal cells is dependent upon infiltrating class II MHC+ CD11bbright monocytic/macrophagic cells.
Authors: Hammerberg C , Duraiswamy N , Cooper K D
Published: January 2023

Contact sensitizers, tumor Ags, and microbial pathogens presented through UV-exposed skin result in T cell-mediated immune tolerance (inhibition of acquisition of responsiveness) to these normally potent immunogens. The APC in UV-exposed skin that delivers the signals inducing tolerance remains highly controversial and is the subject of this study. Application of the contact sensitizer, 2,4-dinitro-1-fluorobenzene (DNFB), to C3H/HeN mice immediately after a single dose of 72 mJ/cm2 UVB (138 mJ/cm2 total UVB) resulted in unresponsiveness to an initial DNFB ear challenge, but failed to block the development of responsiveness after a second sensitization on previously unexposed skin (no tolerance). A state of tolerance could only be achieved if a delay of 72 h was allowed to elapse between the UV exposure and the initial sensitization. Epidermal cell suspensions (EC) were prepared from the skin of normal controls and from skin exposed to the same UV dose 3 days before (UV-EC). Three days after in vivo UV exposure, Langerhans cells (CD11blow Ia+) were depleted, and CD11bbright Ia+ macrophages had appeared in the epidermis along with GR-1+ neutrophils. Intracutaneous injection of 2,4 dinitrobenzenesulfonic acid (DNBSO3)-haptenated UV-EC, but not normal controls, resulted in the induction of locally inducible Ag-specific tolerance to DNFB, indicating the presence and dominance of tolerogenic signal within in vivo-irradiated epidermis. Removal of CD11b+ and class II MHC+ cells within UV-EC showed that a CD11b+ class II MHC+ population was indeed critical for tolerance induction. In addition, tolerance induction by UV-EC was not a result of surviving, UV-exposed Langerhans cells, because haptenated 3-day cultured EC from epidermis removed 5 h after UV exposure (before leukocytic infiltration) failed to induce a tolerogenic state. In conclusion, the ability of UV-exposed skin to induce peripheral adult tolerance to a normally potent immunogen is critically dependent on inflammatory class II MHC+, CD11bbright monocytic/macrophagic cells that infiltrate UV-irradiated skin at the same time the ability to tolerize is acquired.
Expression of recombinase-activating genes (RAG-1 and 2) in human decidual mononuclear cells.
Authors: Hayakawa S , Saito S , Nemoto N , Chishima F , Akiyama K , Shiraishi H , Hayakawa J , Karasaki-Suzuki M , Fujii K T , Ichijo M
Published: January 2023

Recombinase-activating genes (RAG-1 and RAG-2) are expressed in immature T or B lymphocytes and possess activity to induce V(D)J rearrangement in TCR and Ig genes. We examined their expression in human decidual and non-pregnant endometrial samples using a highly sensitive RT-PCR technique. Expression of RAG-1 and 2 was noted in all (13/13) pregnant decidual tissues obtained at different gestational stages. After anti-human Ig treatment to rule out the possibility of RAG-1,2 expression from decidual B-cells, strong expression of RAGs was still noted in B cell depleted decidual cells in contrast to lymph nodes and PBMC that lost RAG mRNA expression after this treatment. After FACS mediated cell sorting, strong expression of RAG-1,2 was noted in CD16-CD56bright cells and weak expression in CD3+ cells. Although CD16-CD56bright cells lack surface CD3, they express CD3 epsilon mRNA only detectable by RT-PCR. Our results suggest the nature of decidual CD16-CD56bright cells as a progenitor of extrathymic T cells that possess RAG-1 and 2 mRNA as markers of their immaturity, and possibly differentiate into CD3+ extrathymic T-cells in the decidua under the influence of trophoblastic cells. We propose the human decidua as a new site of extrathymic T-cells differentiation and propose possible roles of trophoblastic cells to attract progenitor lymphocytes of bone marrow origin and trigger their TCR rearrangement as thymic epithelial cells.
Expression of CD43 on murine and human pluripotent hematopoietic stem cells.
Authors: Moore T , Huang S , Terstappen L W , Bennett M , Kumar V
Published: January 2023

To understand regulation of hemopoiesis, it would be helpful to identify physiologically relevant function-associated molecules on stem cells. Here, we report the detailed examination of CD43 expression on murine and human pluripotent hemopoietic stem cells. Mouse stem cells were found within the Ly6+Lin-CD43high subpopulation of bone marrow. These cells, upon transfer into SCID mice, caused rapid repopulation of thymus, spleen, and bone marrow. Retransfer of bone marrow cells from primary SCID recipients of Ly6+Lin-CD43high cells into secondary recipients resulted in repopulation of lymphohemopoietic cells. All Ly6+Lin-CD43high cells were found to express high levels of c-kit. In contrast, Ly6+Lin-CD43-/low cells caused limited and variable thymic and splenic repopulation. These cells failed to repopulate the marrow cavity and did not contain retransplantable stem cells. These data indicate that murine pluripotent stem cells express high levels of CD43. Examination of human fetal bone marrow cells revealed a population of CD34+CD38-CD43+ cells. When single sorted cells with this phenotype were cultured in vitro, they were able to produce colonies with a dispersed growth pattern. Cells with this growth pattern have previously been shown to have myeloid and lymphoid growth potentials and extensive self-renewal capacity. Furthermore, CD34+CD38-HLA-DR+ cells, recently shown to be highly enriched in stem cell activity, expressed relatively high levels of CD43. Because CD43 has recently been shown to bind to intercellular adhesion molecule-1, these data suggest a possible role for CD43 in the regulation of hemopoiesis.
The mechanism of autoantibody production in an autoimmune MRL/lpr mouse.
Authors: Shan H , Shlomchik M J , Marshak-Rothstein A , Pisetsky D S , Litwin S , Weigert M G
Published: January 2023

Rheumatoid factors (RF) and anti-DNA Abs from MRL/lpr mice have features similar to Abs directed toward foreign Ags, indicating a role of specific activation by Ags during disease. But our previous studies and analogous studies from others concentrated on a limited subset of hybridomas selected on the basis of Ag binding to well characterized target autoantigens. Thus, it has been unclear to what extent clonal expansion is restricted to identifiable autospecificities. To obtain a more complete picture of disease-associated autoantibody production, we designed the following experiment. A large number of B cell hybridomas were generated from the spleen of an MRL/lpr mouse and then analyzed for self-specificity, sequence, and clonal relationship. Surprisingly, we found that clonal expansion was limited to only a few autospecificities, implying a unique property of this response. In addition, we used Southern blotting with heavy and L chain constant region probes to screen both RF and non-RF hybridomas for membership in clones, one of which was first identified among RF hybridomas. We found no non-RF members of this clone. The size and number of mutations of this clone were sufficient for us to conclude that nonspecific (i.e., non-RF) mutant members are rapidly lost. Had an Ag other than IgG2a been driving clonal expansion, we should have seen mutants that retained spectificity for that Ag but that lost specificity for IgG2a. This observation, along with the restriction of clonal expansion to a few autospecificities, provides strong evidence that normal autoantigens themselves drive autoantibody clonal expansion.
Proteolytic activity of an antibody light chain.
Authors: Sun M , Gao Q S , Li L , Paul S
Published: January 2023

The light chain (L chain) of a mAb raised against unactivated vasoactive intestinal peptide (VIP) hydrolyzed this peptide, whereas the heavy chain (H chain) and an irrelevant L chain were without activity. The reaction kinetics were consistent with efficient substrate recognition by the anti-VIP L chain compared with conventional proteases. The L chain cleaved four peptide bonds clustered between residues 16 and 21 in VIP. Mixtures of the L chain with its H chain partner displayed reduced hydrolytic activity compared with the free L chain, suggesting that the H chain is a modulator of the catalytic activity. These observations suggest: 1) the immune system can generate catalytic sites in the L chain subunit of Abs found in response to polypeptide Ags, and 2) free L chains found in vivo could display an Ag-specific catalytic function.
Abnormal signal transduction by T cells of mice with parental tumors is not seen in mice bearing IL-2-secreting tumors.
Authors: Salvadori S , Gansbacher B , Pizzimenti A M , Zier K S
Published: January 2023

There is considerable evidence to demonstrate that immune function is abnormal in tumor-bearing mice, perhaps accounting, at least in part, for progressive tumor growth. In an attempt to generate an antitumor response, we used retroviral vectors to express IL-2 cDNA in CMS5, a murine fibrosarcoma. Mice inoculated with unmodified tumor cells suffered progressive tumor growth, whereas tumors secreting IL-2 were rejected or grew slowly. Animals bearing unmodified but not IL-2-secreting tumors also were immunosuppressed. On the basis of these observations, we were interested in how IL-2 secretion by the tumor cells prevented the onset of hyporesponsiveness. To identify biochemical differences between T cells of mice with parental vs slowly growing IL-2-secreting tumors, we examined signal transduction after activation through the CD3/TCR complex. Protein tyrosine phosphorylation was altered and calcium flux was reduced in cells of mice with parental tumors compared with animals with slowly growing IL-2-secreting tumors. In addition, levels of protein for the tyrosine kinases p56lck and p59fyn, as well as the TCR-zeta-chain, were reduced. These differences in signal transduction were observed for T cells of mice with parental and IL-2-secreting tumors of the same size, demonstrating that differences in tumor size alone could not explain our findings. Thus, IL-2 secretion by tumors seems to be able to prevent immunosuppression by maintaining normal signal transduction in T cells, facilitating the generation of antitumor responses.
Protective cytotoxic T lymphocytes are induced during murine infection with Chlamydia trachomatis.
Authors: Starnbach M N , Bevan M J , Lampe M F
Published: January 2023

T cell responses are often an important component in immunity to organisms that replicate intracellularly. Cytotoxic T lymphocyte (CTL) recognition of peptide Ag in the context of MHC class I molecules results in lysis of infected cells and the release of cytokines including IFN-gamma. Members of the genus Chlamydia are obligate intracellular pathogens that cause blindness and sexually transmitted disease worldwide. Even though it replicates within a membrane-bound vacuole, Chlamydia trachomatis may elicit a CTL response if Chlamydia Ags are present in the cytoplasmic compartment where they can be processed for presentation and bound by MHC class I. In this study, we characterized a CTL line derived from mice infected with C. trachomatis. This CTL line is specific for, and able to lyse, Chlamydia-infected cells. The peptide epitope recognized by this CTL line is present on infected cells, and is presented to the CTL by the classical MHC class I molecule H-2 Ld. Adoptive transfer of this CTL line into an infected mouse affords protection, and this protection requires the activity of IFN-gamma.
Acyclic analogue of lipid A stimulates TNF-alpha and arachidonate release via a unique LPS-signaling pathway.
Authors: Fagan M A , Liu Y , Stütz P , Vyplel H , Golenbock D T
Published: January 2023

LPS has been implicated in the pathogenesis of Gram-negative bacterial sepsis. Despite intensive efforts to define the LPS-signal transduction pathway, CD14 is the sole molecule clearly demonstrated to possess signaling capabilities. However, it remains unclear whether CD14 is the only LPS-signaling molecule expressed in phagocytes and how CD14-mediated signaling occurs. Compound SDZ 280.961 is a synthetic triacylated amino acid that structurally resembles the reducing sugar moiety of lipid A. SDZ 280.961 effectively stimulated TNF-alpha release from human PBMC. Co-incubation of PBMC with the specific LPS inhibitor Rhodobacter sphaeroides lipid A inhibited SDZ 280.961-mediated stimulation of TNF-alpha release, indicating that this analogue signals mononuclear cells via a LPS-activated signaling pathway. Induction of TNF-alpha release from mononuclear cells by SDZ 280.961 was strongly dependent on the presence of serum and was enabled by the presence of purified LPS-binding protein, characteristics of CD14-mediated signaling. In contrast, SDZ 280.961-mediated signaling was not inhibited by blocking anti-CD14 mAbs. A Chinese hamster ovary fibroblast line transfected with human CD14, which responds to LPS in a manner qualitatively similar to that of macrophage cell lines, failed to respond to SDZ 280.961. Taken together, these data suggest that the lipid A analogue SDZ 280.961 activates monocytes via a unique LPS-signal transduction pathway that appears to be independent of CD14.
CD11b+ macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL-10 and represent the major secretory source of epidermal IL-10 protein.
Authors: Kang K , Hammerberg C , Meunier L , Cooper K D
Published: January 2023

Because activated human macrophages can be potent sources of IL-10, and because immunosuppressive tolerance-inducing macrophages populate the skin after UV exposure, we determined whether IL-10 is induced after UV exposure of human skin and whether it is related to the immigrating macrophages. Keratomes were obtained from control skin or from skin obtained 72 h after a single exposure to four minimal erythemal doses of UVB. Quantitative reverse-transcriptase PCR on total RNA extracted immediately from skin keratomes showed that IL-10 mRNA was elevated in UV-exposed skin. Epidermal cell suspensions from non-UV-exposed keratomes (C-EC) and UV-exposed keratomes (UV-EC) were fractionated by sequential immunobead selection. IL-10 mRNA was reproducibly 200- to 400-fold higher in CD11b+ UV-EC (macrophages) relative to CD11b- UV-EC (keratinocytes). IL-10 mRNA was not detected in C-EC that contained the CD1a+ population (Langerhans cells) nor in CD1a- C-EC keratinocytes from normal skin. As determined by ELISA, CD11b+ UV-EC IL-10 cell-associated protein was fivefold higher than that of CD11b- UV-EC; this was confirmed by flow cytometric visualization of IL-10 protein in permeabilized cells. CD11b+ UV-EC macrophages secreted IL-10 protein into the supernatant at a level of 333 +/- 51 pg/10(6) cells, whereas UV-EC keratinocytes did not secrete detectable levels of IL-10 (n = 3), although UV did induce low levels of IL-10 mRNA and cell-associated protein in keratinocytes. Therefore, although human keratinocytes accumulate intracellular IL-10 after in vivo UV exposure, the most potent production and secretion of IL-10 in the epidermis seems to be that of UV-induced macrophages. Skin-infiltrating macrophage secretion of such a potent immunoregulatory cytokine may account for the delayed immunosuppressive environment of sunburned skin and the altered APC activity of the infiltrating macrophages.
Differential responses of fibroblasts from wild-type and TNF-R55-deficient mice to mouse and human TNF-alpha activation.
Authors: Mackay F , Rothe J , Bluethmann H , Loetscher H , Lesslauer W
Published: January 2023

The role of the two TNF receptor types, TNF-R55 and TNF-R75, was studied on mouse fibroblasts, taking advantage of TNF-R55-deficient mice generated by gene targeting (Tnfr1 degree-mice), and selectivity of human TNF-alpha for mouse TNF-R55. Radioligand binding assays showed that both TNF receptors were expressed on wild-type mouse fibroblasts, whereas normal levels of TNF-R75 were expressed on mouse fibroblasts isolated from Tnfr1 degree-mice. It was found that TNF-R55 controlled four major TNF-induced fibroblast functions: (1) adhesion to leukocyte cell lines as well as ICAM-1, VCAM-1, CD44, and MHC class I up-regulation; (2) secretion of other cytokines as demonstrated by stimulated IL-6 and granulocyte-macrophage-CSF releases; (3) cell proliferation; and (4) NF-kappa B activation. Stimulation through TNF-R75, in TNF-R55-deficient fibroblasts, did not have any effect in these functions. In general, mouse TNF-alpha (recognizing both mouse TNF receptors) had a higher sp. act. than human TNF-alpha (recognizing only mouse TNF-R55) in wild-type fibroblasts, whereas both mouse and human TNF-alpha had similar cytotoxic activities in WEHI 164 cells.
Interactions of human complement component C3 with factor B and with complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) involve an acidic sequence at the N-terminus of C3 alpha'-chain.
Authors: Taniguchi-Sidle A , Isenman D E
Published: January 2023

Complement component C3 is a multifunctional protein that interacts with many different ligands and receptors. Several experimental approaches involving blocking Abs, proteolytic fragmentation, and synthetic peptides have been used to predict which regions in C3 are required for its various functions. We have used site-directed mutagenesis to alter specific residues in the C3 segments 730-739 and 933-942 that have been proposed to be required for the binding of factor B by C3, and have examined, within the context of the intact C3b molecule, the effect of these substitutions on the C3b-factor B interaction. Because it has been suggested that factor H and complement receptors type 1, 2, and 3 may recognize sites in C3 that partially or completely overlap those of factor B, the relevant proteolytic fragment of each mutant C3 was tested for its ability to interact with these molecules. This study clearly demonstrates that a segment near the N-terminus of the alpha'-chain, which contains the negatively charged residues 730DE and 736EE, is involved in the interactions of C3 proteolytic fragments with factor B and complement receptors type 1 and 3, but not type 2. Factor H cofactor activity was also partially affected by these mutations. In contrast, mutation of the 937KED triplet in the C3 933-942 segment had little or no effect on any of these activities.
C1q binding properties of monomer and polymer forms of mouse IgM mu-chain variants. Pro544Gly and Pro434Ala.
Authors: Taylor B , Wright J F , Arya S , Isenman D E , Shulman M J , Painter R H
Published: January 2023

The effect of replacing proline with alanine at position 434 in the C mu 3 domain (P434A) and with glycine at position 544 in the C mu 4 domain (P544G) of the mu-chain of mouse IgM has been studied. The P434A substitution results in the loss of measurable complement-mediated cytolytic activity (CML) and a decrease in the association rate constant at low ionic strength (mu = 0.06), that results in a diminished Ka for C1q binding to P434A IgM bound to haptenated cells (0.4 x 10(9) M-1). Binding of C1(qr2s2) could not be detected. In contrast, replacement of proline at 544 had no measurable effect on the cytolytic or C1q/C1 binding properties of the polymeric molecule, supporting the view that the C mu 3 domain is important in C1q binding and CML. The secreted monomeric subunit of P544G was not able to mediate CML. Also, whereas hapten-bound P544G polymer bound C1q with a functional affinity of 1.5 x 10(9) M-1 at low ionic strength (mu = 0.06), similar to that observed with wild-type polymer (1.7 x 10(9) M-1) and wild-type IgG monomer (4.7 x 10(9) M-1), no C1q binding was detected with the P544G IgM monomer. This could not be attributed to differences in glycosylation. Inasmuch as the P544G mutation per se had no effect on the C1q binding properties of the polymer, we conclude that unlike IgG, aggregation does not sufficiently enhance the avidity of IgM monomer to enable it to activate complement. Augmentation of the site must occur during polymerization or when the IgM binds to Ag.
Effect of MHC class I and CD8 cell deficiency on experimental autoimmune myasthenia gravis pathogenesis.
Authors: Shenoy M , Kaul R , Goluszko E , David C , Christadoss P
Published: January 2023

MHC class I and CD8+ cell deficiency have either prevented systemic lupus erythematosus-like disease in mice or enhanced type I diabetes in nonobese diabetic mice. To study the involvement of MHC class I and class I-restricted CD8+ T cells in the induction of a classical Ab-mediated disease, experimental autoimmune myasthenia gravis (EAMG), we immunized beta 2 microglobulin (beta 2-m) gene-disrupted (beta 2 m-/-) C57BL10 (B10) mice, deficient in class I gene expression and CD8+ cells, and heterozygous (beta 2-m+/-) B10 mice with normal expression of class I molecules and sufficient CD8+ cells with Torpedo acetylcholine receptor in CFA, and assessed them for clinical and immunopathologic manifestations of EAMG. Despite MHC class I and CD8+ cell deficiency, beta 2-m-/- mice developed EAMG. Moreover, the incidence of EAMG in the beta 2-m-/- mice was higher than that of beta 2-m+/- heterozygous mice with normal class I expression and frequency of CD8+ cells. The finding provided direct genetic evidence against a pathogenic effector role in C57BL10 mice for MHC class I molecule and class I-restricted CD8+ T cells in EAMG pathogenesis.
TNF-alpha associated with fibronectin enhances phorbol myristate acetate- or antigen-mediated integrin-dependent adhesion of CD4+ T cells via protein tyrosine phosphorylation.
Authors: Hershkoviz R , Cahalon L , Miron S , Alon R , Sapir T , Akiyama S K , Yamada K M , Lider O
Published: January 2023

The effects of cytokines on immune cells may be influenced by their milieu, such as the extracellular matrix (ECM), in the vicinities of which cytokines and inflammatory cells interact and function. Previously, we demonstrated that TNF-alpha bound to fibronectin (FN) and augments the level of adhesion of activated CD4+ cells. Herein, we examined the mechanisms of this pro-adhesive activity of TNF-alpha and its putative physiologic consequences using human or rat CD4+ cells. A brief exposure of CD4+ cells to low dosages of soluble TNF-alpha or of FN- or laminin-bound TNF-alpha synergized with PMA to enhance the integrin-mediated binding of CD4+ cells to these immobilized ECM moieties. TNF-alpha-enhanced adhesion of CD4+ cells did not delay or inhibit the subsequent detachment of the cells from the substrate, and adhesion was increased provided the cells were treated with TNF-alpha immediately after their exposure to PMA. This indicates that the enhancing effect of TNF-alpha requires a previous activation of the cells. When TNF-alpha was immobilized on FN, less TNF-alpha was required to induce CD4+ cell binding to FN. Soluble, and to a greater extent FN-bound, TNF-alpha synergizes with PMA to intensify protein tyrosine phosphorylation in FN-bound CD4+ cells, and this effect of TNF-alpha was inhibited by inhibitors of tyrosine kinase. That FN-bound or soluble TNF-alpha also amplified the binding of an Ag-specific autoimmune rat T cell line to immobilized FN, emphasizes the physiologic relevance of our findings. Thus, the signal transduction and cell adhesive properties of ECM glycoproteins may be modulated upon their association with TNF-alpha, and matrix-linked TNF-alpha may recruit and direct immune cells to inflammatory sites.