bluuebunny/crossref_metadata_2025 huggingface dataset:
Extensive allelic polymorphism in the CDR2-like region of the miniature swine CD4 molecule.
Authors: Gustafsson K , Germana S , Sundt T M , Sachs D H , LeGuern C
Published: December 2022

MHC class II polymorphism is well documented whereas only minimal polymorphism has been reported for the CD4 molecule with which it interacts. We report on the structural basis of an allelic polymorphism of the CD4 molecule in miniature swine. Eleven of 13 nucleotide differences between the 2 alleles cause amino acid replacements. A majority of these replacements are clustered in a region protruding as a loop structure, termed Ig CDR2-like, from the surface of the amino terminal domain. This part of the human CD4 appears to comprise the binding site for the human immunodeficiency virus gp120 protein. The loop structure has also been implicated in the binding of CD4 to MHC class II, but this is currently a matter of some controversy. Our previous results have indicated that the porcine CD4 polymorphism, that we now show is situated in this loop, does not significantly affect the binding to class II. However, because the polymorphism appears to have been selected for and is situated in a very exposed part of the molecule, it is likely to be of functional significance.
Evolving T cell responses in murine schistosomiasis. Th2 cells mediate secondary granulomatous hypersensitivity and are regulated by CD8+ T cells in vivo.
Authors: Chensue S W , Warmington K S , Hershey S D , Terebuh P D , Othman M , Kunkel S L
Published: December 2022

The dynamics of T cell maturation and regulation were examined during the granulomatous response to Schistosoma mansoni eggs under conditions of primary (PRIM), secondary vigorous (VIG), and secondary immunomodulated (MOD) immunity. The patterns of VIG and MOD GR (GR) formation were established in naive CBA mice by transfer of lymphoid cells from S. mansoni-infected donors at the VIG and MOD stages of infection. Sequential production of IFN, IL-2, and IL-4 was assessed at the GR and in draining lymph nodes (LN) to determine the participation of Th1 (IFN-producing) and Th2 (IL-4/IL-5-producing) cells. The PRIM GR produced IFN in the growth phase (2-16 days) and IL-4 was detected only after lesions were established (16-24 days). The PRIM LN showed coincident production of IFN, IL-2, and IL-4 on day 4 followed by mainly IL-4 and IL-2 production on day 8, consistent with Th2 differentiation via a Th0 precursor. The VIG GR produced high levels of IL-4 in the growth phase (4-8 days) although IFN remained at modest levels. The VIG LN showed increased cytokine levels consistent with an anamnestic response and again the pattern suggested Th2 differentiation from Th0 cells. The MOD mice had abrogated GR IL-4 levels and arrested Th2 differentiation in LN. In vitro mix studies indicated that the impaired cytokine production was not due to direct suppression. The role of Ts cells was next explored by T cell subset depletion. Pan-T cell depletion of VIG cells profoundly abrogated IL-4 production although CD8+ cell depletion augmented GR area by 70% as well as local and regional IL-4 production by 100 to 150%. Similarly, CD8+ cell depletion of MOD cells augmented GR size and IL-4 production but the response was less than corresponding VIG mice, suggesting that Th activity was reduced in MOD mice. Transfer studies indicated that CD8+ cells inhibited Th2 maturation in LN. Thus, the schistosome egg GR demonstrated a Th1-like pattern in the PRIM response followed by a Th2 pattern in the VIG stage and abrogated Th2 activity in the MOD stage. Finally, the phenomenon of "spontaneous modulation" likely represents the cumulative action of CD8+ cells which steadily erode the Th population.
IL-4 release by human leukemic and activated normal basophils.
Authors: Arock M , Merle-Béral H , Dugas B , Ouaaz F , Le Goff L , Vouldoukis I , Mencia-Huerta J M , Schmitt C , Leblond-Missenard V , Debre P
Published: December 2022

Recently, authors have addressed the ability of human basophils to produce IL-4. We report here the detection of significant serum IL-4 levels in a case of acute transformation of chronic myelogenous leukemia with a predominant basophilic cell population. Leukemic basophils were isolated from patients' PBMC and assayed for their IL-4-mRNA expression and their ability to secrete this cytokine in vitro. Leukemic basophilic cells (> 90% toluidine blue positive) but not other PBMC expressed IL-4-mRNA, contained IL-4 protein, and secreted this cytokine. These cells had a spontaneous IL-4 secretion ability, without a need for an exogenous activator. Meanwhile, IL-4 release was significantly increased following leukemic cell activation through Fc epsilon RI-ligation or by Ca2+ ionophore. IL-4 and its mRNA were also detected in leukemic basophils from three other chronic myelogenous leukemia patients with moderate basophilia (13, 14, and 23% basophils in PBMC). To confirm these data in normal human cells, we have developed a method to obtain large numbers of purified basophils from human bone marrow cell cultures. In contrast to leukemic basophils, normal cells required in vitro activation through Fc epsilon RI ligation or by Ca2+ ionophore to express and secrete IL-4. Leukemic and normal basophils secreted histamine following in vitro activation, but were negative for tryptase. These data thus demonstrate the in vivo and in vitro ability of human basophils to produce IL-4.
Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-gamma, and IL-3.
Authors: Gosselin E J , Wardwell K , Rigby W F , Guyre P M
Published: December 2022

Polymorphonuclear neutrophils (PMN) have long been thought to be short-lived, terminally differentiated cells incapable of synthesizing significant levels of protein, with their primary function being phagocytosis and the release of cytotoxic compounds. More recently, it has been demonstrated that PMN can produce a number of functionally diverse substances, including IL-1, IL-6, and IL-8. Although PMN express class I MHC Ag, it has not been definitely demonstrated that they can synthesize and express class II Ag. This would suggest that, although PMN can indirectly assist in the induction of an immune response through production of cytokines, they are incapable of acting as APC for CD4+ Th cells. We show that, in the presence of a defined medium (AIM V), human serum, and granulocyte-CSF, nearly 100% of isolated PMN can survive for up to 2 days in vitro. We also show that PMN express MHC class II when present as bystander cells in a monocyte/T cell Ag presentation assay for 44 h. In addition, granulocyte/macrophage CSF (GM-CSF), IFN-gamma, and IL-3 can induce class II on pure cultures of PMN, with GM-CSF appearing to be the most potent of the three cytokines. Furthermore, induction of class II on PMN is distinctly donor dependent, with PMN from some donors repeatedly showing very high, and others very low, induction of class II when treated with GM-CSF. Their potential to express class II suggests that PMN could play a significant role in immunoregulation and disease pathogenesis. The variation in class II induction on PMN from individual donors might explain previous failures to detect class II induction on PMN and could be a factor in the varied susceptibility of different individuals to autoimmune and inflammatory disorders such as the production of antibodies to PMN cytoplasmic components.
Characterization of the immunosuppressive effects of nitric oxide in graft vs host disease.
Authors: Hoffman R A , Langrehr J M , Wren S M , Dull K E , Ildstad S T , McCarthy S A , Simmons R L
Published: December 2022

The generation of nitric oxide (.N = O) during in vitro assays involving lymphocyte-macrophage interaction can result in profound inhibition of lymphocyte proliferation. The present study examined whether .N = O synthesis plays a role in the suppression observed in immune function assays during graft vs host disease (GvHD). By using a parent to F1 model to induce GvHD (C57BL/6J to C57BL/6J x DBA 2J F1), a mild but transient increase in serum NO2- plus NO3- levels was observed on day 12 after inoculation. Resident peritoneal macrophages obtained from mice with GvHD demonstrated enhanced .N = O synthesis in response to LPS, compared with control F1 peritoneal macrophages. Similarly, when splenocytes from GvHD mice were cultured with Con A or LPS enhanced supernatant NO2- levels were observed, compared with control F1 mice. Addition of NG-monomethyl-L-arginine (NMA), a competitive inhibitor of .N = O synthesis, resulted in decreased NO2- levels and greatly enhanced proliferation in response to Con A. Addition of NMA to LPS-stimulated cultures did not enhance proliferation, perhaps as the result of the paucity of B cells in the GvHD population. LPS-induced .N = O synthesis by GvHD splenocytes was blocked by anti-IFN-gamma mAb, whereas Con A-induced .N = O synthesis was relatively unaffected by similar concentrations of anti-IFN-gamma mAb, suggesting different mechanisms of induction of .N = O synthesis. A proliferative response of splenocytes from mice with GvHD to third-party alloantigen was not detectable, even in the presence of NMA. The suppression observed when splenocytes from GvHD animals were added to control TNP-modified self cultures was partially reversed in the presence of NMA. These results demonstrate that .N = O synthesis in both splenocyte and peritoneal macrophage populations from GvHD mice is enhanced, revealing that in vivo priming of macrophages for .N = O synthesis occurs during GvHD. Some, but not all, in vitro tests of immune function by using GvHD splenocytes are suppressed by the generation of .N = O.
Membrane cofactor protein (CD46) protects cells predominantly from alternative complement pathway-mediated C3-fragment deposition and cytolysis.
Authors: Kojima A , Iwata K , Seya T , Matsumoto M , Ariga H , Atkinson J P , Nagasawa S
Published: December 2022

Membrane cofactor protein (MCP) cDNA was transfected into Chinese hamster ovarian tumor (CHO) cells and the functional properties of the expressed protein were studied. Cells adherent to flasks were essential for continuous expression of MCP on CHO cells. If the cells were maintained in noncoated flasks, MCP expression was markedly reduced but upon being transferred to coated flasks reexpressed the protein. MCP expressed on CHO cells had the expected m.w., approximately 50 kDa, and possessed factor I-cofactor activity. By a propidium iodide incorporation assay and by 51Cr release assay, antibody-sensitized CHO cells expressing MCP were protected from C-mediated cytotoxicity. The inhibition of lytic activity correlated with a decrease in C3 deposition. This host cell protective activity was exerted efficiently for the alternative pathway. The classical pathway was not blocked by MCP unless the cells were presensitized with low concentrations of antibody. These results imply that MCP primarily protects host cells from alternative pathway-mediated C3 targeting. In a pathologic state such as autoimmune diseases, the binding of an autoantibody to a target may overcome this protective effect of MCP via the classical pathway.
Regulation of expression of the ligand for CD40 on T helper lymphocytes.
Authors: Castle B E , Kishimoto K , Stearns C , Brown M L , Kehry M R
Published: December 2022

Activated Th cells deliver contact-dependent signals to resting B lymphocytes that initiate and drive B cell proliferation. Recently, a ligand for the B lymphocyte membrane protein, CD40, has been identified that delivers contact-dependent Th cell signals to B cells. A dimeric soluble form of CD40 was produced and used to further characterize the regulation of expression of the CD40 ligand. Expression of the CD40 ligand was rapidly induced after Th lymphocyte activation, and its stability depended upon whether Th cells were activated with soluble or plastic-bound stimuli. Th cells activated with soluble stimuli rapidly turned over cell-surface CD40 ligand whereas Th cells activated with plastic-bound stimuli exhibited more stable CD40 ligand expression for up to 48 h. Removal of activated Th cells from the plastic-bound stimulus resulted in a rapid turnover of CD40 ligand, suggesting that continuous stimulation could maintain CD40 ligand expression. Ligation by soluble CD40 could also stabilize expression of CD40 ligand on the Th cell surface. Both CD40 ligand and IL-2 were transiently synthesized from 1 to 12 h after Th cell activation and had similar kinetics of synthesis. In Con A-activated Th cells newly synthesized CD40 ligand exhibited an initial high turnover (1.5 h t1/2) and after 5 h of Th cell activation became more stable (10-h t1/2). In Th cells activated with plastic-bound anti-CD3, CD40 ligand exhibited a similar biphasic turnover except that the rapid turnover phase began significantly later. This delay could allow more time for newly synthesized CD40 ligand to assemble or associate with other molecules and thus become stabilized on the cell surface. Newly synthesized CD40 ligand in Con A-activated Th cells appeared to not be efficient in delivering Th cell-dependent contact signals to resting B cells, implying the need for assembly or accessory proteins. Regulation of CD40 ligand expression was consistent with all the characteristics of Th cell-delivered contact signals to B cells and may contribute to the high degree of specificity in B cell responses.
Expression of merosin in the thymus and its interaction with thymocytes.
Authors: Chang A C , Wadsworth S , Coligan J E
Published: December 2022

Merosin is a 700 kDa multichain protein that has several properties in common with laminin, one of the major extracellular matrix proteins present in basement membranes. Both contain B1 and B2 light chains, but merosin has a distinct M-chain compared with the laminin A-chain. Merosin is present in the basement membrane of placenta, striated muscle, and peripheral nerve. Using the RT-PCR assay, we demonstrate that merosin, but not laminin, is also expressed in the thymus. A partial cDNA sequence of the mouse merosin M-chain was 88% identical to the human M-chain, and the deduced amino acid sequences were 95% identical. 18-24% of adult mouse thymocytes bound to human merosin in vitro. Mg2+, but not Ca2+, was essential for binding. Binding was inhibited by antibodies recognizing VLA alpha 6 or beta 1, suggesting that the VLA-6 integrin is a merosin receptor. An anti-beta 4 integrin subunit mAb failed to inhibit binding, suggesting that the alpha 6 beta 4 integrin was not involved. Thymocytes were fractionated into immature and mature populations based on their expression of the heat stable antigen, recognized by the J11d mAb. Virtually all thymocytes expressed VLA-6, but only immature thymocytes (J11d+) bound to meroson. PMA treatment did not significantly increase the binding of J11d+ thymocytes nor did induce binding in the mature J11d- population. In constrast, both splenic T cells and unseparated lymph node cells showed enhanced binding to merosin after PMA stimulation. The expression of merosin in the thymus and its selective interaction with immature thymocytes suggest that thymocyte-merosin interactions may play a role in T cell development.
Role of CD4+ and CD8+ cell subsets during amplification of natural T cell activity against IgG2ab in Igha mice and during induction of IgG2ab allotype suppression in Igha/b mice.
Authors: Majlessi L , Benaroch P , Denoyelle C , Bordenave G
Published: December 2022

Transfer into F1 Igha/b mice of splenocytes from Igha mice sensitized once against B cells from an Ighb congenic strain induces total, chronic, and IgG2ab (IgG2a of the Ighb haplotype)-specific allotype suppression in these recipients. We previously demonstrated that both the CD4+ and CD8+ T subsets were necessary for inducing suppression, but that CD8+, cells by themselves were sufficient for maintaining suppression. We have studied the suppression induction capacity of different mixtures of CD4+ and CD8+ subsets obtained by in vitro cytotoxic treatment of T splenocytes from normal or sensitized Igha mice, and we have established that suppression induction requires the cooperation between CD4+ and CD8+ populations, both of which have to be IgG2ab specific. In addition, Igha mice were sensitized in the absence of CD4+ or CD8+ cells by in vivo cytotoxic treatment performed before and after the sensitization in order to obtain an IgG2ab-specific CD4+ population that has arisen in the absence of CD8+ cells, and vice versa. We found that only IgG2ab-specific CD4+ cells from anti-CD8-treated mice (T'sens CD4+) had the ability to induce suppression in F1 Igha/b hosts. Nevertheless, the real effector cells in this suppression model display the CD8+ phenotype, as in vivo cytotoxic anti-CD8 treatment of Igha/b recipients of T'sens CD4+ abrogates the suppression induction capacity. Taken together, these results show that T'sens CD4+ have an important capacity to recruit CD8+ anti-IgG2ab effector cells from precursors that have been transferred with them into Igha/b hosts. These precursors are actually derived from the T'sens CD4+ cell preparation, because we have recently demonstrated that suppression is maintained by donor T cells throughout the recipient's life. CD4+ cells can have their anti-IgG2ab activity amplified only by means of target cells (i.e., B cells from Ighb congenic mice), whereas, in the absence of CD4+ cells, and despite the presence of target cells, CD8+ cells seem unable to acquire this amplified activity.
Two signals are required for negative selection of CD4+CD8+ thymocytes.
Authors: Page D M , Kane L P , Allison J P , Hedrick S M
Published: December 2022

Recent results indicate that two signals are required for activation of mature T cells. The first is delivered through the TCR, and the second is delivered through receptors that bind various ligands expressed on APC. For example, it has been shown that B7/BB1, which is expressed on many APC, can costimulate T cell activation by binding to CD28 or CTLA4, which are expressed on mature T cells. In contrast, little is known of the signals required for negative selection of autoreactive thymocytes. Thus, we have investigated this issue by using an in vitro culture system in which thymocytes from mice that are transgenic for a class II MHC-restricted TCR are cultured with murine fibroblast lines that express class II MHC. Under these conditions, CD4+CD8+ (DP) thymocytes undergo an Ag-dependent programmed cell death, which likely represents the negative selection of autoreactive thymocytes that would occur in an intact thymus. Using this culture system, we first found that both TCR- and APC-dependent stimuli were required in order to induce deletion of DP thymocytes. Anti-TCR antibodies alone did not cause deletion of DP cells, but merely induced a decrease in their expression of CD4 and CD8 to produce a DPdull phenotype. Addition of APC was then required for deletion of these DPdull cells. One obvious candidate for the costimulatory signal expressed by these APC was B7. Three different experimental approaches indicated, however, that B7 was not the APC-dependent signal required for deletion of DP thymocytes. Thus, these results suggest that negative selection of autoreactive thymocytes is a two-step process in which stimulation of the TCR causes downregulation of CD4 and CD8 on DP thymocytes, and then an unknown ligand expressed on APC stimulates a receptor on DP thymocytes to induce their deletion.
Specific V beta T cell subsets mediate the immediate hypersensitivity response to ragweed allergen.
Authors: Renz H , Saloga J , Bradley K L , Loader J E , Greenstein J L , Larsen G , Gelfand E W
Published: December 2022

T and B cell responses after sensitization to ragweed (RW) were examined in a mouse model in which BALB/c mice were exposed to the allergen by ultrasonic nebulization. Sensitization resulted in the stimulation of an IgE anti-RW response and was paralleled by a rise in IgG1 anti-RW titers. Skin testing for immediate cutaneous hypersensitivity revealed the presence of allergic type I reactions to RW. Sensitization to RW in this way was also associated with the development of increased airways responsiveness as determined by electrical field stimulation of preparations of tracheal smooth muscle. Histologic examination of the airways and the lung indicated the presence of a mononuclear cell infiltrate in the mucosa and submucosa of the airways that was accompanied by an enlargement of local draining lymph nodes of the airways and the lung. T cell populations were analyzed for the frequency of V beta-expressing T cells. Such analysis indicated that RW sensitization stimulated the expression of V beta 8.1+, V beta 8.2+, and V beta 13+ T cells in the local lymphoid tissue and of V beta 8.1+, V beta 8.2+, V beta 8.3+, V beta 9+ and V beta 14+ T cells in the spleen. Co-culture of these T cell populations with RW-primed B cells indicated that in the presence of RW, V beta 8.2 T cells stimulated IgE and IgG1 production, whereas the other T cell populations showed a different stimulation profile for Ig isotypes and IgG subclasses. The transfer of V beta 8.2 T cells from sensitized but not from nonsensitized control mice stimulated an allergen-specific IgE and IgG1 response and increased airways responsiveness in naive recipients. These data provide additional support for the pivotal role of specific V beta-expressing T cell subpopulations in the stimulation of IgE/IgG1 production and increased airways responsiveness.
Anti-TAPA-1 antibodies induce protein tyrosine phosphorylation that is prevented by increasing intracellular thiol levels.
Authors: Schick M R , Nguyen V Q , Levy S
Published: December 2022

We studied the signal induced by the anti-TAPA-1 antibody and compared it to the signal induced by anti-IgM antibodies in a human B cell line, OCl-LY8. We found that exposure of these cells to either antibody resulted in a rapid increase in protein tyrosine phosphorylation which was prevented by inhibitors of tyrosine kinases. Tyrosine phosphorylation was an early event in the cascase leading to the antiproliferative effect of the anti-TAPA-1 antibody. However, 2-ME, a reducing agent that is not an inhibitor of tyrosine kinases, prevented both tyrosine phosphorylation and the antiproliferative effect of the antibody. Cells grown in low concentrations of 2-ME did not exhibit an increase in tyrosine phosphorylation in response to the anti-TAPA-1 antibody and were insensitive to the antiproliferative effect of the antibody. In contrast, the same cells maintained in 2-ME were able to induce tyrosine phosphorylation in response to anti-IgM. The use of 2-ME resulted in an increase in intracellular thiols, mostly glutathione. Moreover, compounds that block glutathione synthesis rendered cells susceptible to the antibody, even in the presence of 2-ME. These experiments demonstrate that tyrosine kinases are involved in propagating the antiproliferative signal initiated by the anti-TAPA-1 antibody and suggest that this signal is dependent upon the level of intracellular thiols.
Antigen presentation capacity of murine macrophages infected with Leishmania amazonensis amastigotes.
Authors: Prina E , Jouanne C , de Souza Lão S , Szabo A , Guillet J G , Antoine J C
Published: December 2022

Leishmania-infected M phi are potential candidates for the presentation of parasite Ag to Leishmania-specific CD4+ T lymphocytes. To assess whether infected cells could function as APC, we examined the ability of bone marrow-derived M phi infected with Leishmania amazonensis amastigotes to stimulate various CD4+, l-Ad- or l-Ed-restricted T-cell hybridomas specific for the bacteriophage lambda repressor cl protein, the human chorionic gonadotropin or OVA. A reduced capacity of infected M phi to present native Ag to most T-cell hybridomas tested was noted that was probably a result of a lower expression on their plasma membrane of stimulatory [la-peptide] complexes. Neither a reduced Ag uptake nor an altered Ag processing appeared to be at the origin of the partial inability of infected M phi to present Ag. As regards the level of plasma membrane la expression, no quantitative difference could be detected between uninfected and infected M phi. Moreover, after fixation with paraformaldehyde, the ability of plasma membrane la molecules to bind immunogenic peptides was apparently not reduced in infected M phi. So, these cells most likely expressed functional la molecules on their cell surface. Interestingly, infected M phi and M phi infected then cured by a treatment with a leishmanicidal compound were similarly impaired in their capacity to present native Ag or peptides to the hybridomas, and no recovery was noted even 24 h after the leishmanicidal treatment. Furthermore, infected M phi and M phi incubated with heat-killed amastigotes or with an amastigote homogenate exhibited similar inhibitions of Ag presentation. Taken together, these results suggest that the functional failure of infected M phi to present exogenous Ag could be because either of interferences with the events leading to the meeting of la molecules with peptides derived from these exogenous Ag or to a competition for binding to la molecules between these peptides and parasite molecules.
Targeted activation of CD8 cells and infection of beta 2-microglobulin-deficient mice fail to confirm a primary protective role for CD8 cells in experimental leishmaniasis.
Authors: Wang Z E , Reiner S L , Hatam F , Heinzel F P , Bouvier J , Turck C W , Locksley R M
Published: December 2022

CD8+ T cells play an important role in the immunologic control of intracellular pathogens, particularly viruses. Leishmania are obligate intracellular parasites of macrophages in the mammalian host, and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells. Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis. In BALB/c mice susceptible to Leishmania major (L. major) infection, targeted activation of CD8+ T cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism, gp63, that contained the consensus binding motif for MHC class I H-2Kd molecules. Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against P815 cells pulsed either with peptide or lysates of L. major. Purified CD8+ T cells from immunized mice had elevated levels of IFN-gamma mRNA transcripts as compared to unimmunized mice. Despite evidence for activation of CD8+ cells, none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, beta 2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored. These mice cured disease as rapidly as beta 2-m +/- and +/+ littermates, and cure was associated with comparable levels of IFN-gamma mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L. major.
Receptor-ligand interactions between serum amyloid P component and model soluble immune complexes.
Authors: Brown M R , Anderson B E
Published: December 2022

We report here that isolated human serum amyloid P (SAP) binds aggregates of human IgG (AAg), which are model soluble immune complexes. The binding interaction was specific with regard to AAg compared to monomeric IgG, saturable, of high affinity, and reversible. With SAP adsorbed to microtiter plate wells, protein binding assays of AAg preparations with m.w. values of 1.05 to 2.25 x 10(6) were performed. The assay results yielded binding isotherms and estimates of the dissociation constant values of SAP:AAg binding ranging between 0.60 and 1.9 nM. Using the same method, the dissociation constant of SAP binding of biotinylated AAg was estimated to be approximately 3.0 nM. Competitive protein binding assays using biotinylated AAg and unlabeled AAg or monomeric IgG were performed to determine the relative affinity (IC50) of SAP binding. The IC50 values ranged from 22 to 51 nM for different sizes of AAg. The IC50 determined for monomeric IgG was 10 microM, demonstrating a relative specificity of SAP binding for AAg. The binding interaction was reversible, the SAP:AAg complexes dissociating with a t1/2 of 125 min. AAg binding to solid phase-adsorbed SAP was not dependent upon the presence of several cations, including calcium, but was not supported by cuprous ion. The differential binding of AAg vs monomeric IgG reported here for SAP is similar to the other human immune complex binding proteins C1q, rheumatoid factor, and cellular Ig C-region receptors. These results indicate that SAP:AAg binding has the hallmarks of a receptor:ligand interaction and may have implications for the disposal of immune complexes in vivo.
Quantitation and kinetics of blood monocyte migration to acute inflammatory reactions, and IL-1 alpha, tumor necrosis factor-alpha, and IFN-gamma.
Authors: Issekutz A C , Issekutz T B
Published: December 2022

Monocytes migrate from the blood into acute inflammatory reactions, where they differentiate into macrophages, and after 12 to 24 h become the predominant histologic feature of the inflammatory infiltrate. The quantitation of monocyte migration into these reactions has been difficult. This report employs a novel combination of techniques to isolate highly purified monocytes from the blood of rats, and shows that these cells have a normal t1/2 of 26 h and migrate efficiently after i.v. injection into cutaneous acute inflammatory sites. Monocytes labeled with 51Cr accumulated in delayed-type hypersensitivity reactions, and sites injected with killed Escherichia coli, LPS, poly-inosine: cytosine, zymosan-activated serum (ZAS), a source of C5adesArg, and the cytokines IL-1 alpha, TNF-alpha, and IFN-gamma. Both radiolabeled monocytes and neutrophils migrated rapidly to E. coli, LPS, ZAS, IL-1 alpha, and TNF-alpha with a large increase in cell accumulation by 2 h. Neutrophil migration declined rapidly to undetectable levels by 3 to 4 h to all five stimuli, and monocyte migration to ZAS and IL-1 alpha also declined by this time. In contrast, E. coli, LPS, and TNF-alpha caused a sustained migration of monocytes for 5 to 6 h, long after neutrophils had stopped accumulating. Intradermal IFN-gamma did not recruit neutrophils but stimulated a prolonged monocyte migration from 1 to 6 h. Combinations of LPS, TNF-alpha, and IFN-gamma synergistically enhanced the late (> 5 h) but not the early phase of monocyte recruitment. In conclusion, purified monocytes isolated from rat blood can be used to quantify monocyte migration in vivo, and these cells migrate rapidly to cutaneous inflammation and in response to chemotactic factors, IL-1 alpha, TNF-alpha, and IFN-gamma, with initial kinetics similar to those of neutrophils. However, monocyte-selective mechanisms are induced by IFN-gamma and also appear to be involved in prolonged monocyte migration to TNF-alpha, LPS, and E. coli.
Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide.
Authors: Merrill J E , Ignarro L J , Sherman M P , Melinek J , Lane T E
Published: December 2022

Rat ameboid microglia are able to lyse rat oligodendrocytes in vitro. The lysis is inhibited by transforming growth factor-beta, antagonists of nitric oxide (NO) production, as well as antibodies to TNF-alpha, intercellular adhesion molecule-1 (ICAM-1), and leukocyte functional Ag-1. Ameboid microglial cells spontaneously produce detectable levels of the NO metabolite nitrite (NO2-). Stimuli such as PMA, LPS, and/or IFN-gamma induce micromolar concentrations of NO2- within 24 h. TNF-alpha increases IFN gamma but not LPS-induced NO2- production. Incubation with target oligodendrocytes also increases NO2- production in a contact-dependent manner. NO2- production is inhibited by NO synthase antagonists, transforming growth factor-beta, and anti TNF-alpha. Neither antileukocyte functional Ag-1 nor anti-ICAM-1 inhibit NO2- production by microglia in the presence or absence of oligodendrocytes. Indeed, anti-ICAM-1 treatment increases NO2- production. There is a correlation between ameboid microglial cell killing of oligodendrocytes and NO2- production suggesting NO may be a mechanism of death of the oligodendrocyte and possibly play a role in lesion formation in multiple sclerosis.
Mechanism of increased tumor necrosis factor production after thermal injury. Altered sensitivity to PGE2 and immunomodulation with indomethacin.
Authors: Molloy R G , O'Riordain M , Holzheimer R , Nestor M , Collins K , Mannick J A , Rodrick M L
Published: December 2022

Altered macrophage function after thermal injury is associated with increased production of PGE2 and TNF. However, it is not clear why synthesis of both cellular products remains elevated, as PGE2 is a potent inhibitor of TNF secretion. We studied the relationship between PGE2 and TNF synthesis in a murine model of thermal injury, and examined the effect of prostaglandin blockade on splenic macrophage secretion of these mediators of inflammation. LPS-stimulated production of PGE2 was significantly elevated in burn groups compared with sham-burned controls (pg/ml mean(SEM); sham 151(32): burn 597(147), p < 0.01). TNF production was similarly increased after thermal injury (pg/ml mean(SEM); sham 62(20): burn 928(316), p < 0.01). In vitro culture of macrophages with indomethacin augmented LPS stimulated TNF production in sham-burned controls but did not affect synthesis in burn groups, suggesting a loss of PGE2-dependent regulation of TNF synthesis after thermal injury. Direct measurement of TNF secretion as a function of exogenous PGE2 confirmed this dissociation between PGE2 and TNF synthesis, as burned animals displayed a 5-fold reduction in sensitivity to PGE2-induced inhibition of TNF, when compared with sham-burned controls (ID50 PGE2 molar; sham 1.26 x 10(-8): burn 6.43 x 10(-8), p < 0.05). In vivo pretreatment of burn groups with indomethacin for 5 days before assay partially restored sensitivity to the prostaglandin, and significantly down-regulated synthesis of both TNF and PGE2. These data show that thermal injury is associated with a loss of PGE2-dependent down-regulation of TNF synthesis, which accounts at least in part for increased TNF in these animals. In vivo cyclooxygenase blockade partially restored sensitivity to the prostaglandin and consequently down-regulated synthesis of TNF. These data further support existing evidence that suggests a potential therapeutic role for cyclooxygenase blockade after major thermal injury and trauma.
Cord blood contains high numbers of autoimmune T cells recognizing multiple myelin proteins and acetylcholine receptor.
Authors: Fredrikson S , Sun J B , Huang W X , Li B L , Olsson T , Link H
Published: December 2022

To analyze Ag-specific T cell autoimmunity in the newborn, umbilical cord blood cells of newborns were investigated by an immunospot assay for occurrence and numbers of cells secreting IFN-gamma after short-term culture in presence of myelin basic protein (MBP), proteolipid protein, myelin associated glycoprotein, nicotinic acetylcholine receptor and the synthetic MBP amino acid sequences 1-20, 63-88, and 110-128. These Ag were chosen because they represent putative targets for autoimmune attack in multiple sclerosis and myasthenia gravis. Surprisingly, numbers of T cells recognizing MBP, proteolipid protein, MBP peptides, and acetylcholine receptor were high in cord blood of newborns compared to peripheral blood of patients with neurologic diseases. No immunodominant T cell epitope could be discerned among the Ag included. The responses to purified protein derivate and PHA were lower among cord blood cells than peripheral blood cells of adults. Parallel enumeration of autoimmune T cells in cord blood and peripheral blood obtained from corresponding mothers at delivery, revealed that the high cell numbers in newborns were not a result of contamination from the mothers blood. The high numbers of T cells recognizing nervous system myelin proteins and acetylcholine receptor in cord blood could have importance for the mechanism and timing of tolerance induction, and also reflect excessive myelination and receptor maturation at birth.
In vivo antigen presentation by both brain parenchymal cells and hematopoietically derived cells during the induction of experimental autoimmune encephalomyelitis.
Authors: Myers K J , Dougherty J P , Ron Y
Published: December 2022

A fundamental issue in the etiology of autoimmune diseases of the central nervous system such as multiple sclerosis and its animal counterpart experimental autoimmune encephalomyelitis (EAE) concerns the identity of cells capable of presenting autoantigen to the T cells that mediate these diseases. The prevailing dogma is that only bone marrow-derived cells function as APC during EAE induction. We have addressed this issue by studying EAE induction in mouse bone marrow chimeras, and have found that although bone marrow-derived APC such as macrophages and brain microglial cells are more efficient at presenting autoantigen, brain parenchymal cells such as astrocytes and endothelial cells are also capable of inducing disease. EAE was induced in these chimeras by the adoptive transfer of encephalitogenic T cell lines designed to be MHC-histocompatible with APC contained either within the hematopoietic system of the chimera or with APC resident to the brain of the chimera. The subsequent development of EAE in these chimeras then indicated which population of cells served as in vivo APC during EAE pathogenesis. Possible effects of alloreactivity between the host chimera and the adoptively transferred T cells were eliminated by using encephalitogenic T cell lines made tolerant to the haplotype(s) of the recipient chimera.
A humanized CD18 antibody can block function without cell destruction.
Authors: Sims M J , Hassal D G , Brett S , Rowan W , Lockyer M J , Angel A , Lewis A P , Hale G , Waldmann H , Crowe J S
Published: December 2022

Leukocyte integrins are intimately involved in transient adherence of leukocytes to endothelium and to each other in the processes of extravasation and cell activation. In this study, seven mAb directed against human CD11a and two mAb directed against human CD18, the alpha- and beta-chains of the leukocyte functional Ag-1 molecule, respectively, were analyzed for their ability to inhibit several leukocyte functional Ag-1-mediated interactions. The best blocking mAb in these studies, a rat anti-human CD18, YFC51.1, was subsequently humanized by complementarily-determining region grafting, associated with human C regions and expressed. The humanized mAb was shown to maintain binding for human CD18. Even though the humanized mAb was an IgG1 isotype it still retained the functional blocking characteristics of the rat mAb while failing to mediate cell killing. The IgG1 mAb was unable to bind human Clq and could block but did not mediate antibody-dependent cellular cytotoxicity.
Modulation of perforin and granzyme messenger RNA expression in human natural killer cells.
Authors: Salcedo T W , Azzoni L , Wolf S F , Perussia B
Published: December 2022

Perforin and granzymes are proteins thought to play a relevant role in cell-mediated cytotoxicity. These molecules are constitutively expressed in NK cells and their level of expression in cytotoxic T lymphocytes is regulated by several cytokines. We analyzed the mechanisms by which cytokines and cellular ligands known to modulate NK cell-mediated cytotoxicity affect the expression of the mRNA encoding granzyme A and B and perforin in NK cells. Our data indicate that IL-2 and IL-12 induce increased accumulation of both perforin and, to a higher degree, granzyme B mRNA. In contrast, binding of target cells or immune complexes up-regulates expression of granzyme B mRNA without altering that of perforin. Results of in situ hybridization experiments confirm that mRNA for both molecules are expressed at low levels in most NK cells, and that both are induced to accumulate by the two cytokines in the majority of the cells. The mechanisms by which IL-2 and IL-12 regulate expression of the two molecules are, in part, distinct: both cytokines increase the transcriptional rate of the encoding genes, whereas only IL-2 acts also at a post-transcriptional level to increase the stability of their mRNA.
Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling.
Authors: Huczko E L , Bodnar W M , Benjamin D , Sakaguchi K , Zhu N Z , Shabanowitz J , Henderson R A , Appella E , Hunt D F , Engelhard V H
Published: December 2022

Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.
Coordinate silencing of myeloma-specific genes in myeloma x T lymphoma hybrids.
Authors: Lieberman S A , Hines M D , Bergsagel P L , Kuehl W M , Eckhardt L A
Published: December 2022

It has been well-established that Ig genes are transcriptionally silenced when Ig-producing myeloma lines are fused to non-B cells. In the present study, we analyzed the expression of several other myeloma-specific genes in fusions of myelomas with the T lymphoma, BW5147. Seven of the eight genes analyzed behaved coordinately with the Ig loci; they were silent in most myeloma x T hybrids but active in the rare hybrid that retained Ig gene expression. Cloned IgH genes introduced into the two types of hybrids behaved as their endogenous counterparts. The coordinate behavior of these several genes in the panel of "exceptional" and "extinguished" hybrids suggests a central and bimodal switch for alternately activating and de-activating the genetic program of the Ig-secreting plasmacyte. The switch between an active and an inactive transcriptional state involves, at some level, a change in the methylation status of the IgH genes. Methylation and transcriptional activity were inversely correlated. In Ig-extinguished hybrids the myeloma-derived locus was methylated de novo, whereas in the rare Ig-expressing hybrid, the T cell-derived locus was demethylated de novo.
IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor.
Authors: Schadendorf D , Möller A , Algermissen B , Worm M , Sticherling M , Czarnetzki B M
Published: December 2022

Normal melanocytes require a number of exogenous growth factors in contrast to most metastatic malignant melanomas. This investigation demonstrates that endogenously produced human IL-8 can act as an important growth factor for human melanoma cells. In the present study, six out of eight human melanoma cell lines tested secrete IL-8 protein into the culture supernatant. In two of these IL-8-secreting melanoma cell lines, SK-MEL 13 and SK-MEL 23, we have determined the IL-8 requirement for their proliferative capacity. These melanoma cell lines produced significant amounts of bioactive IL-8 as measured by the ELISA technique. Secretion of human IL-8 was inducible by IL-1 and by PMA. Human IL-8-specific mRNA was already detected in unstimulated melanoma cells. In addition, human IL-8-R mRNA could be detected for the first time in human melanoma cells. Exposure of the two melanoma cell lines in vitro to antisense oligonucleotides targeted against two different sites of human IL-8 mRNA-inhibited cell proliferation, colony formation in soft agar, and secretion of IL-8 protein into culture supernatant in a dose dependent fashion. Effects were reversible either by removal of the oligomers or by addition of exogenous IL-8 protein. In contrast, exposure to IL-8 sense probes or oligonucleotides in sense or antisense orientation specific for IL-7, TGF-alpha, TGF-beta, and MGSA had no such effect. A monospecific immune serum and two IL-8-specific mAb were also capable of inhibiting melanoma cell proliferation in the same manner. These results provide strong evidence for an autocrine IL-8 synthesis and for an IL-8-dependent proliferation in a subgroup of human melanomas. Furthermore, they suggest that IL-8 may play a role not only in immunomodulation but also in melanoma progression and metastatic spread.
Epitope mapping and functional analysis of three murine IgG1 monoclonal antibodies to human tumor necrosis factor-alpha.
Authors: Bloom J W , Bettencourt J D , Mitra G
Published: December 2022

Immunologic and enzyme digest epitope mapping techniques were used to compare and contrast the interactions of three mouse mAb, designated A10G10, A6, and B6, with human TNF-alpha. These antibodies have previously been shown to protect mouse and human cells from TNF toxicity. ELISA showed that the antibodies recognize predominantly conformational epitopes, and that native TNF binds at least two molecules of the same mAb. Enzyme digestion of native TNF bound to each of the mAb in turn showed that each of the antibodies binds to or sterically masks a large fraction of the exposed surface of TNF. These epitope mapping data were compared with four putative TNF cell receptor binding sites presented in the literature. The results are consistent with the hypothesis that these antibodies prevent TNF-induced cell death by binding to or sterically masking the TNF receptor binding site.
Epitopes of streptococcal M proteins that evoke antibodies that cross-react with human brain.
Authors: Bronze M S , Dale J B
Published: December 2022

There is evidence suggesting that Sydenham's chorea, which is a major manifestation of acute rheumatic fever, may be mediated by streptococcal antibodies that cross-react with the brain. Our studies were undertaken to determine whether streptococcal M protein, the major virulence factor of group A streptococci, evoked antibodies that cross-react with human brain. Rabbits were immunized with pepsin-extracted M protein from rheumatogenic type 6 streptococci. Immune sera were screened for the presence of antibodies that cross-reacted with human brain by indirect immunofluorescence tests and immunoblot analyses. Type 6 M protein evoked antibodies that cross-reacted with several brain proteins and antibody binding to these proteins was completely inhibited by type 6 M protein and partially inhibited by types 5 and 19 M proteins, suggesting that these heterologous M proteins contain conserved brain-cross-reactive epitopes. Using synthetic peptides from several serotypes of M proteins, the conserved brain-cross-reactive epitopes were localized to a decapeptide contained within the covalent structure of the B repeat region of type 6 M protein. These peptides also inhibited brain-cross-reactive antibodies in the serum of a patient with active Sydenham's chorea. Our data indicate that streptococcal M proteins contain brain-cross-reactive epitopes that could potentially be involved in the pathogenesis of Sydenham's chorea.