bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Fc mu receptor on human natural killer cells. Interaction with its physiologic ligand, human normal IgM, specificity of binding, and functional effects.
Authors: Pricop L , Rabinowich H , Morel P A , Sulica A , Whiteside T L , Herberman R B
Published: December 2022

After treatment with human normal IgM, 78 +/- 8% of purified CD3-CD56+ resting human NK cells and 93 +/- 6% of IL-2-activated NK cells selected by adherence to plastic reacted with FITC-goat anti-human IgM. Binding of IgM to the FcR for IgM (Fc mu R) on human NK cells was not species specific because mouse myeloma IgM also bound to these cells. The percentage of CD56+ cells binding IgM after incubation with anti-CD16 mAb was similar to that of cells incubated with medium alone (95 +/- 1% vs 93 +/- 4%). Binding of anti-CD16 mAb to Fc gamma RIII on NK cells was unaffected by pretreatment with IgM (65 +/- 12% vs 69 +/- 4%). The CD7 molecule has been reported to be the Fc mu R on the surface of T cells. Two-color flow cytometry showed that 94 +/- 3% of CD3-CD56+ resting NK cells and 71 +/- 16% of activated NK cells were CD7+. Preincubation of NK cells with three anti-CD7 mAb (Leu-9, 8H8-1, and LAU-A1) failed to block the binding of IgM to the Fc mu R. Modulation of the CD7 molecule off the cell surface (CD7+ = 1.5% +/- 0.3) did not reduce IgM binding, thus excluding the possibility that IgM anti-CD7 might bind to different epitopes of the same molecule. These data indicate that the Fc mu R is a specific Ig-binding structure, distinct from the Fc gamma RIII (CD16) or CD7. The Fc mu R on NK cells functions as a signal-transducing molecule because the addition of 0.2 mg/ml IgM to R-NK cells caused a rapid increase in [Ca2+]i (delta = 40 nM). One of the early events that followed signaling through the Fc mu R was the down-modulation of IFN-gamma gene expression and IFN gamma production in NK cells. The presence of IgM during culture of NK cells consistently decreased the expression of HLA-DR (16% vs 40% in control). Thus, the Fc mu R, a constitutively-expressed receptor on human NK cells, seems to be an important functional molecule, which delivers negative regulatory signals to NK cells.
High occupancy binding of antigenic peptides to purified, immunoadsorbed H-2Db beta 2m molecules.
Authors: Burshtyn D N , Barber B H
Published: December 2022

In an effort to examine the peptide binding properties of purified class I MHC molecules, we have developed a solid phase, radiolabeled peptide binding assay based on the use of H-2Db molecules bound to agarose beads via heavy chain-specific mAb. Using purified Db beta 2m, recovered from RMA-S cells and bound to immunoadsorbent beads through either alpha 1 or alpha 3 region specific antibodies, complete occupancy of these molecules could be achieved with 125I-Y366-374 influenza nucleoprotein peptide (Kd 10(-7) M). Approximately 12% of the Db beta 2m dimers recovered from RMA cells could be occupied by this influenza nucleoprotein peptide under the same conditions. When free Db heavy chains were isolated from beta 2m negative R1E.Db cells by bead-bound alpha 3-region specific antibody (28-14-8S) and were incubated with human beta 2m, high affinity (Kd 10(-8) M) binding sites were created for the 125I-Y367-374 influenza nucleoprotein peptide. In addition to demonstrating that a significant fraction of the heavy chains present in R1E.Db cells are in a beta 2m-reactive form, the R1E.Db cells provide an alternate approach to that of RMA-S derived Db beta 2m empties for the creation of homogeneous complexes of Db, beta 2m, and antigenic peptide. We anticipate that these bead-bound empty and defined peptide-class I complexes may be useful in the further study of class I MHC target structure formation and recognition.
Dynamics of peptide binding to purified antibody-bound H-2Db and H-2Db beta 2m complexes.
Authors: Burshtyn D N , Barber B H
Published: December 2022

Although it is clear that each component of the class I MHC trimolecular complex (heavy chain, beta 2m, and antigenic peptide) contributes to its formation and stability, the specific interaction governing assembly and disassembly remain to be clarified. In an effort to address these issues using purified H-2Db molecules, we used a solid-phase binding assay recently developed in our laboratory to quantify kinetic parameters for class I assembly and disassembly. It was found that the influenza NP peptide Y367-374 associated with preformed empty complexes of 28-14-8S- (i.e., anti-alpha 3) bound Db beta 2m dimers much more quickly (t 1/2 < 0.2 h at 22 degrees C) than it did when coincubated with an anti-alpha 3 bound Db and human beta 2m (t1/2 3.5 h at 22 degrees C). The previously reported potential for the NP peptide Y367-374 to interact directly with free Db heavy chains and configure the conventionally beta 2m-dependent B22 epitope in the absence of beta 2m, was confirmed using our assay system. However, the rate of B22 epitope formation induced in the Db heavy chain by NP Y367-374 was considerably slower (t1/2 13 h, at 22 degrees C) and much less efficient on a molar basis than that resulting from the addition of beta2m (t1/2, 0.75 h, at 22 degrees C). In contrast, the Db heavy chain with NP-Y367-374 was more resistant to thermal disassembly (as measured by loss of the B22 epitope, t1/2 2h, 37 degrees C) than the Db beta 2m empty dimer (t1/2 0.2 h). Finally, stability of the preformed trimolecular complex of heavy chain, beta 2m, and peptide was found to diminish in accordance with deviation of the peptide from the optimal length and with increasing temperature from 4 to 37 degrees C.
Simultaneous involvement of all six predicted antigen binding loops of the T cell receptor in recognition of the MHC/antigenic peptide complex.
Authors: Kasibhatla S , Nalefski E A , Rao A
Published: December 2022

The TCR is predicted to resemble the Fab fragment of an Ig molecule and by analogy to possess six Ag binding loops that contact MHC proteins bound with antigenic peptides. We have identified residues in the predicted Ag binding loops (beta 1, beta 2, and beta 3) on a TCR beta-chain that are important in the recognition of the MHC/antigenic peptide complex. Using site-directed mutagenesis, we altered the residues forming the predicted Ag binding site on the beta-chain expressed by the T lymphocyte clone D5, which specifically recognizes p-azobenzenearsonate-conjugated peptides presented by the class II MHC molecule I-Ad. Amino acid substitution of individual residues in each loop affected Ag recognition, demonstrating that all three putative Ag binding loops of the D5 TCR beta-chain are important in interaction with I-Ad/arsonate-conjugated Ag. Taken together with our previous work on the D5 TCR alpha-chain (Nalefski et al., J. Exp. Med. 175:1553), these results suggest that all six Ag binding loops of the D5 TCR alpha- and beta-chains interact simultaneously with the MHC/peptide complex. Consequently, the area of interaction between the TCR and the MHC/antigenic peptide complex is predicted to be extensive.
Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase.
Authors: Arnholdt A C , Piuvezam M R , Russo D M , Lima A P , Pedrosa R C , Reed S G , Scharfstein J
Published: December 2022

Human infection with Trypanosoma cruzi (Chagas' disease) is usually accompanied by humoral and cellular immune responses to GP57/51, a major antigen that was recently identified as a prominent cysteinyl proteinase (cruzipain). The PBMC responses of 11 chronic chagasic patients and the properties of anti-cruzipain T cell lines are reported herein. GP57/51, isolated from Y strain epimastigotes (n-cruzipain) or the recombinant protein expressed in E. coli (r-cruzipain), elicited proliferative responses of variable intensity from the patient's PBMC. T cell lines were then generated using each of these antigens. These lines, which always carried the CD4+ phenotype, were reciprocally stimulated by n-cruzipain or r-cruzipain, the responses to the former being usually stronger. The analysis of cytokine production suggested that Th1-like subsets dominate the patient's responses: IFN-gamma was consistently induced on stimulation with either n-cruzipain or r-cruzipain. In contrast, IL-4 was present in very small concentrations or was undetectable. We then sought to define T cell epitopes of cruzipain using synthetic peptides spanning portions of the central (catalytic) domain and COOH-terminal extension. From a panel of 11 peptides, only one 33 mer peptide (P214) elicited a strong proliferative response on anti-cruzipain T cell lines, the intensity being comparable to that induced by r-cruzipain. Conversely, T cell lines started with P214 were responsive to either n-cruzipain or r-cruzipain, the proliferative responses again being accompanied by IFN-gamma production, but not IL-4. Interestingly, P214 is located in a conserved region of the catalytic domain of cruzipain, hence may propitiate opportunities for cross-recognition of other members of the papain superfamily. Fine epitope mapping should reveal whether structurally similar regions of host thiol-cathepsins can be potential targets for cross-reactive T cell responses during chronic human infection.
Reversible expression of mouse mast cell protease 2 mRNA and protein in cultured mast cells exposed to IL-10.
Authors: Ghildyal N , Friend D S , Nicodemus C F , Austen K F , Stevens R L
Published: December 2022

BALB/cJ mouse mast cells derived by culturing bone marrow progenitor cells in WEHI-3 cell-conditioned medium (BMMCW) do not contain mouse mast cell protease 2 (mMCP-2) mRNA, but these cells can be induced to express this transcript after exposure to rIL-10. To study the translation and granule accumulation of mMCP-2 in rIL-10-treated BMMC (BMMCW+IL-10), a rabbit antibody was developed to a synthetic peptide that corresponds to the novel amino acid sequence in mMCP-2 at residues 56 to 71. After affinity purification, this antibody, anti-mMCP-2(56-71) IgG, reacted in SDS-PAGE/immunoblots against a 28-kDa protein in BMMCW+IL-10 that had the N-terminal amino acid sequence of mMCP-2. As assessed immunohistochemically, mMCP-2 protein accumulated in the secretory granules of Kirsten sarcoma virus-immortalized mouse mast cells, BMMCW+IL-10, and the mucosal mast cells present in the jejunum of Trichinella spiralis-infected BALB/cJ mice. Time course analyses of the induction of mMCP-2 mRNA and protein in BMMCW+IL-10 revealed that these cells contain a high steady-state level of mMCP-2 mRNA 24 h after their exposure to rIL-10. Although a small amount of immunodetectable mMCP-2 protein is present in the cells treated for 24 h, large amounts of this protease are not obtained until 7 days of treatment of the cells with rIL-10. Time course analyses of the loss of mMCP-2 mRNA and protein in BMMCW+IL-10 revealed that the steady-state level of mMCP-2 mRNA decreased dramatically 24 h after rIL-10 was removed from the culture medium, but that the level of mMCP-2 protein did not decline measurably until day 5 of culture. The fact that the steady-state levels of mMCP-2 mRNA and protein in BMMC can both be reversibly altered by culturing these mast cells in the presence and absence of rIL-10 suggests that the phenotype of mast cells is not fixed. Rather, it is in a dynamic state regulated by the cytokine network to which mast cells are exposed in their different microenvironments.
Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysaccharide on free attractants, specific IgG autoantibodies and immune complexes.
Authors: Sylvester I , Suffredini A F , Boujoukos A J , Martich G D , Danner R L , Yoshimura T , Leonard E J
Published: December 2022

We recently found that normal human sera contain IgG antibodies against two chemoattractants, neutrophil attractant protein-1 (NAP-1/IL-8) and monocyte chemoattractant protein-1 (MCP-1), as well as immune complexes of these proteins. Intravenously administered LPS was reported to cause a sharp rise in serum NAP-1 concentration. Our study was designed to determine if LPS also caused an increase in MCP-1 and to measure associated changes in concentrations of antibody and immune complex. LPS caused a rise to peak within 2 to 3 h in serum concentrations of free NAP-1 and MCP-1, followed by an almost equally rapid fall toward base-line levels by about 5 h postinjection. MCP-1 concentration in sera from the 11 subjects rose to a peak of 330 +/- 52 pM. The peak value for NAP-1 was 80 +/- 11 pM. In 10 of the 11 subjects, free IgG autoantibody to MCP-1 decreased from a mean pre-LPS value of 1820 +/- 660 pM to a mean low of 53% of the respective initial values. Corresponding data for IgG anti-NAP-1 were a pre-LPS concentration of 216 +/- 7 pM, which decreased to a mean low of 44% of the respective initial values. The finding in some subjects of a rapid rise in free antibody after the nadir suggests the possibility of acute regulation of autoantibody secretion rates. Although the results suggested that LPS-induced chemoattractant combined with free antibody, serum concentrations of MCP-1-IgG or NAP-1-IgG did not increase, which points to an as yet unknown mechanism for trapping and elimination of the immune complexes.
Synovial fluid macrophages and blood monocytes differ in their response to IL-4.
Authors: Hart P H , Ahern M J , Jones C A , Jones K L , Smith M D , Finlay-Jones J J
Published: December 2022

IL-4 has been described as a potential anti-inflammatory molecule because of its ability in vitro to down-regulate human monocyte production of proinflammatory mediators. In this study, the activity of IL-4 on mononuclear cells and CD14+ macrophages from a site of chronic inflammation, namely, the joints of patients with rheumatoid and psoriatic arthritis, was investigated and compared directly with its activity on PBMC and monocytes from the same patients. In contrast to the response by blood monocytes, the response to IL-4 by synovial fluid cells was selective; IL-4 did not significantly suppress LPS-induced TNF-alpha production, but decreased CD14 expression to a similar extent in the two cell populations. IL-4 induction of monocyte/macrophage CD23 expression was investigated as a stimulatory response to IL-4, and although significantly increased on synovial fluid CD14+ cells by IL-4, the induction was considerably reduced compared with that measured for blood cells. Activation and differentiation in vitro of blood monocytes reduced their response to IL-4 for decreased TNF-alpha production and induction of CD23 and suggested that these biologic phenomena may contribute to the decreased responsiveness to IL-4 by synovial fluid monocytes/macrophages. Thus, IL-4 does not have the same anti-inflammatory properties in vitro on synovial fluid cells as on blood monocytes.
Characterization of CTLA-4 structure and expression on human T cells.
Authors: Lindsten T , Lee K P , Harris E S , Petryniak B , Craighead N , Reynolds P J , Lombard D B , Freeman G J , Nadler L M , Gray G S
Published: December 2022

CTLA-4 is an adhesion receptor expressed on activated T cells. The amino acid sequence of CTLA-4 is related to CD28, and although the function of CTLA-4 remains unknown, it shares several features with CD28, including a common counter-receptor, B7, that is present on Ag-presenting cells. In a recent study we found that CD28 and CTLA-4 were coexpressed at the mRNA level on activated T cells but that only CD28 was expressed on resting T cells. Here we show that within the T cell population, CTLA-4 expression is restricted to the subset of T cells that also express cell surface CD28. CTLA-4 mRNA expression can be induced on quiescent T cells via phorbol ester-mediated activation of protein kinase C but not with calcium ionophore treatment alone. Phorbol ester-induced expression of CTLA-4 mRNA could be enhanced with calcium ionophore treatment, and treatment of cells in this manner resulted in a reciprocal decrease in expression of CD28 mRNA. Ligation of CD28 with monoclonal antibody also resulted in the specific and rapid induction of CTLA-4 mRNA. To study the expression of CTLA-4 at the protein level, a rabbit antiserum against a recombinant protein derived from CTLA-4 cDNA was generated. When activated T cells were labeled with [35S]methionine, the rabbit antiserum precipitated a 41- to 43-kDa protein from whole cell lysates. Similar results were found when detergent-soluble lysates from 125I surface-labeled resting and activated T cells were analyzed by SDS-PAGE. Surprisingly, under the conditions tested, CTLA-4 migrated primarily as a monomer at the cell surface, and could not be shown to exist as a disulfide-bonded homodimer or as a heterodimer consisting of CTLA-4 and CD28. These results suggest that B7 can bind to T cells via distinct receptor complexes consisting of either CD28 or CTLA-4, and that these complexes may potentially mediate distinct biologic functions. Further, the present results suggest that noncovalent interactions might mediate association of CTLA-4 and/or CD28 at the cell surface.
Differential role of conserved and polymorphic residues of the binding groove of MHC class I molecules in the selection of peptides.
Authors: Abastado J P , Casrouge A , Kourilsky P
Published: December 2022

A set of 17-point mutants of H-2Kd was produced as secreted single-chain MHC molecules in the yeast Kluyveromyces lactis. Using a library of 648 synthetic peptides displaying the Kd-specific motif, the repertoire of peptides selected by each mutant was compared by a two-dimensional analysis technique. When conserved residues of Kd were substituted by an alanine, no binding was observed. When polymorphic residues were changed, two outcomes were observed. For residues 95, 99, and 116, no binding was observed, implying that the side chains of these residues contribute directly to the interaction with the peptide. When positions 9, 45, 97, and 114 were changed to alanine, the repertoire of selected peptides was enlarged. Position 97 was also changed to all four possible residues found in natural variants of mouse MHC class I molecules. The repertoires of selected peptides were analyzed and appeared to be included one in another. Their dimension was inversely correlated with the size of the side-chain of residue 97. We conclude that during the course of evolution some polymorphic residues may have been selected for their capacity to reduce the size of the peptide repertoire by preventing certain peptides from binding.
Heavy chain joining region segments of the channel catfish. Genomic organization and phylogenetic implications.
Authors: Hayman J R , Ghaffari S H , Lobb C J
Published: December 2022

The JH locus of the channel catfish has been characterized to determine the organization and structural diversity of JH segments. These analyses indicate that there are a total of nine JH segments tightly clustered within a region spanning about 2.2 kb. The JH locus is closely linked to the CH 1 domain of the expressed catfish H chain; the distance between the CH proximal JH segment (JH9) and the CH 1 domain is about 1.8 kb. Each JH segment has an upstream recombination sequence, which includes a T-rich nonamer, a 22- to 24-bp spacer, and a phylogenetically conserved heptamer. Each JH segment also has an open reading frame that encodes the conserved framework region 4 tryptophan (Trp-103) and terminates with a RNA donor splice site. The catfish JH locus contains an internal repetitive sequence region characterized by a short (183-188 bp) repeat that occurs sequentially five times. Strong sequence homology as well as the unified length of the repeated sequences indicate that JH segments JH3-JH7 probably arose as the result of a series of homologous but unequal crossover events. Sequence alignments of the duplicated JH segments indicates that there is diversity within the 5-11 nucleotides located immediately downstream from the heptamer, an observation which indicates that closely related JH segments can serve to enhance CDR3 diversity in the expressed H chain. Comparisons of the genomic JH sequences with different cDNA clones indicate that each JH segment is probably functional and that junctional diversity serves an important role in the generation of CDR3 diversity. In addition, single base differences observed in comparisons of JH-encoded regions indicate that there is probably somatic mutation or allelic variation of genomic JH segments. These studies suggest that the characteristic structure and organizational pattern of JH segments in higher vertebrates may have evolved early in vertebrate phylogeny at the level of the bony fish.
Structural analysis of the VH-D-JH segments of human polyreactive IgG mAb. Evidence for somatic selection.
Authors: Ikematsu H , Kasaian M T , Schettino E W , Casali P
Published: December 2022

Polyreactive (natural) antibodies are primarily IgM and account for a major proportion of circulating Ig in humans. They use various V gene segments, in general, in germ line (unmutated) configuration. To analyze the VH regions of polyreactive antibodies, with particular attention at their somatically mutated status, we generated five IgG (three IgG1 and two IgG3) mAb (using B cells from a healthy subject, a patient with insulin-dependent diabetes mellitus and a patient with SLE), which bound with various efficiencies a number of different self and foreign Ag. Gene cloning experiments showed that the VH region sequences were unique to each IgG mAb. The H chain complementary determining region (CDR3) of two IgG (mAb10 and mAb426.4.2F20) displayed an identical stretch of five amino acids (RFLEW), but the other three IgG mAb CDR3 were divergent in both length and composition. The VH gene sequences of two IgG, mAb426.4.2F20 and mAb410.7.F91, were 99% identical to those of the germ line VH4.11 and VH4.21 genes, respectively. Those of the remaining three IgG mAb displayed a number of differences (93.6 to 95.9% identity) when compared with the germ line VH4.18, VH4.11, and hv1263 gene sequences. These and the VH4.21 gene have been found to encode polyreactive IgM and IgA and, in mutated configuration, monoreactive high affinity autoantibodies and antibodies induced by foreign Ag. When compared with the respective framework region, the CDR of three IgG mAb VH segment sequences displayed a significantly higher: 1) frequency of total nucleotide differences (6.1 x 10(-2) vs 4.5 x 10(-2) difference/base); 2) frequency of putative nucleotide changes yielding amino acid replacements (5.6 x 10(-2) vs 1.4 x 10(-2) replacement change/base); and 3) ratio of overall putative replacement to silent (R:S) mutations (11.0 vs 0.4). Thus, the distribution and nature of the nucleotide differences were consistent with a process of somatic mutation and Ag-dependent clonal selection. This was formally proved in IgG mAb426.12.3F1.4 and IgG mAb10 by differentially targeted polymerase chain reaction amplification and cloning and sequencing of the germ line genes that gave rise to the expressed VH segments, using DNA from polymorphonuclear cells of the same subjects whose B cells were used for the generation of these IgG mAb. Somatic mutations might have been responsible for bringing about polyreactivity in originally monoreactive antibodies or, more likely, they accumulated in originally polyreactive antibodies, which after undergoing a process of Ag selection, retained polyreactivity and may have or may have not acquired a higher affinity for the selecting Ag.
Cellular distribution of HLA-G mRNA in transgenic mouse placentas.
Authors: Yelavarthi K K , Schmidt C M , Ehlenfeldt R G , Orr H T , Hunt J S
Published: December 2022

The human MHC class I gene, HLA-G, is unique among members of the class I gene family in that it is nonpolymorphic, and expression is primarily restricted to extraembryonic tissues. To examine regulatory elements that direct tissue- and cell lineage-specific expression of this gene, transgenic mice expressing HLA-G have been established. In this study, in situ hybridization was used to evaluate the cellular distribution of HLA-G mRNA in transgenic placentas. Extraembryonic tissues were obtained at gestation day 12.5 from embryos that had been microinjected with either 6.0 or 5.7 kb of HLA-G genomic DNA and had been transferred into pseudopregnant HLA-G transgenic mice or Swiss mice. The 6.0 kb transgene contained an additional 250 bp at the extreme 5'-end of the upstream region. Genotype of the recipient had no discernable effect on the cellular distribution of HLA-G mRNA. HLA-G mRNA was present in both trophoblast and mesenchymal cells in transgenic placentas carrying 6.0 kb of genomic HLA-G, a pattern strikingly similar to that of HLA-G message distribution in early gestation human placentas. By contrast, in placentas from embryos carrying 5.7 kb HLA-G DNA, specific mRNA was found primarily in mesenchymal cells at the base of the placenta. Thus, the 6.0 genomic fragment contains elements capable of directing HLA-G expression in placentas, and is particularly influential in the trophoblastic cell lineage.
The hemopoietic progenitor 32DCl3(G) cells synthesize C3 molecules and acquire C3b acceptor sites upon differentiation or neoplastic transformation.
Authors: Di Renzo L , Zicari A , Longo A , Realacci M , Naso G , Pontieri G , Lipari M
Published: December 2022

32DCl3(G) cells are a diploid, nontumorigenic, IL-3-dependent hemopoietic progenitor cell line, which undergoes terminal differentiation into neutrophilic granulocytes when cultured in presence of G-CSF. The infection with BALB-Moloney murine sarcoma virus, containing a v-HA-ras oncogene, renders this cell line IL-3 independent and continuously growing in the presence of G-CSF, nontumorigenic and with an apparent block at the level of promyelocyte/myelocyte (32D-Ha-ras). After infection with Abelson murine leukemia virus containing a v-abl oncogene, the cell line originates (32D-abl) that is also IL-3 independent but is tumorigenic and unable to differentiate in the presence of G-CSF. This cellular model allowed us to study the relationship between distinct steps of cell differentiation, neoplastic transformation, and C3 synthesis, activation, and characteristics of binding. We demonstrated that C3 synthesis, release, and cleavage are properties already present in the progenitor 32DCl3(G) cells. The more differentiated 32D-Ha-ras cells acquired C3 acceptor sites, apparently completely saturated by the constitutively released molecules. The transformation with Abelson murine leukemia virus, although it did not affect all these properties, let the cells bind considerable amounts of C3-related fragments activated in normal murine serum through the alternative pathway. This last event was a result of the acquisition of PMSF-sensitive serine proteases associated with the plasma membrane.
Enhanced production of LPS-induced cytokines during differentiation of human monocytes to macrophages. Role of LPS receptors.
Authors: Gessani S , Testa U , Varano B , Di Marzio P , Borghi P , Conti L , Barberi T , Tritarelli E , Martucci R , Seripa D
Published: December 2022

In vitro cultivated human monocytes isolated from normal peripheral blood show a time-dependent differentiation into macrophages characterized by an increased expression of transferrin receptors, CD11/CD18, and CD14 Ag. We measured the secretion of TNF-alpha and IL-6 in freshly isolated monocytes and in differentiated macrophages after LPS treatment. Differentiated macrophages produced significantly higher amounts of TNF-alpha and IL-6 than freshly isolated monocytes. This increased secretion was not a result of an enhanced accumulation of TNF-alpha and IL-6 mRNA, as comparative levels of these transcripts were found in both cell types after LPS treatment. Furthermore, LPS did not induce an antiviral state to VSV3 in monocytes, but it reduced by 3 to 5 log10 the virus yield in differentiated macrophages. The addition of antibodies to IFN-beta completely inhibited the LPS-induced antiviral state to VSV, but antibodies to IFN-alpha, TNF-alpha, or IL-6 were ineffective. A marked accumulation of IFN-beta mRNA was found in both cell types after LPS treatment. Binding experiments with FITC-LPS revealed a slightly higher overall binding affinity for LPS in freshly explanted monocytes as compared with differentiated macrophages, even though the maximal binding was higher in macrophages. In both cell types, the LPS binding was partially inhibited by antibodies to CD14. These results demonstrate that: 1) in vitro differentiation of human monocytes to macrophages leads to an enhanced LPS response in terms of (a) progressive increase of IL-6/TNF-alpha production and (b) acquisition of an IFN-beta mediated antiviral state; 2) this enhanced response to LPS, largely CD14-independent, is not linked to any increased accumulation of cytokine mRNA, but is probably a result of an increased synthesis and/or secretion of these cytokines.
IL-3-dependent murine mast cells undergo apoptosis on removal of IL-3. Prevention of apoptosis by c-kit ligand.
Authors: Mekori Y A , Oh C K , Metcalfe D D
Published: December 2022

It is well established that mast cell proliferation and maturation are regulated by two principle cytokines, IL-3 and the c-kit ligand stem cell factor (SCF). Little is known, however, how these two processes are negatively regulated and thus, how mast cell number is controlled in normal or pathologic processes. In this study we hypothesized that IL-3-dependent mast cells would undergo programmed cell death (apoptosis) on removal of IL-3 as was shown with other growth factor-dependent hemopoietic cells. Apoptotic changes were analyzed using light microscopy, fluorescent staining with acridine orange, flow cytometric analysis, and DNA electrophoresis. We could demonstrate that elimination of IL-3 from either primary bone marrow-derived cultured mast cell cultures (BMCMC) or from the growth factor-dependent mast cell line MCP5 resulted in the characteristic changes of apoptosis including condensed chromatin, fragmented nuclei, cellular vacuolization, typical pattern of propidium iodide or Hoechst 33342 uptake by flow cytometry, and the characteristic 200 bp "ladder" pattern of DNA cleavage. These events were prevented by SCF, an action that was in part mediated by tyrosine kinases, in that the tyrosine kinase inhibitor herbimycin inhibited the action of SCF in preventing apoptosis in IL-3-deprived cells. By using anti c-kit mAb and IL-3-dependent BMCMC obtained from W/Wv mice homozygous for mutation at the w locus that encodes the c-kit receptor, we could also show that SCF exerted its effect via c-kit. Neither dexamethasone nor cyclosporin A inhibited the "rescue" effect of SCF, suggesting that "rescue" was mediated by SCF and not through the induction of other cytokines. Thus, IL-3-dependent mast cells undergo apoptosis on removal of IL-3, an event that is prevented by the addition of SCF through its ligand c-kit, thus demonstrating how these principle mast cell growth factors may act in concert to regulate mast cell number under physiologic conditions.
Immunolocalization of cytokines in the nasal mucosa of normal and perennial rhinitic subjects. The mast cell as a source of IL-4, IL-5, and IL-6 in human allergic mucosal inflammation.
Authors: Bradding P , Feather I H , Wilson S , Bardin P G , Heusser C H , Holgate S T , Howarth P H
Published: December 2022

Allergic mucosal inflammation is characterized by the presence of cell infiltration, predominantly with IgE-sensitized mast cells and activated eosinophils, and appears to be regulated by the local production and release of several cytokines, particularly IL-4 and IL-5. Although attention has focused on the Th2 subpopulation of CD4+ T lymphocytes as an important source of these cytokines, human mast cells have been shown to both store and secrete IL-4 and TNF-alpha. To investigate the expression of cytokines relevant to allergic inflammation and to identify their cellular localization within the nasal mucosa, we have undertaken specific immunohistochemical staining of thin sections of inferior turbinate biopsies from patients with perennial allergic rhinitis and, for comparison, from nonatopic healthy volunteers. The cytokines investigated were IL-4, IL-5, IL-6, and IL-8. In both the normal and rhinitic biopsies numerous cells immunoreactive for IL-4, IL-5, and IL-6 were seen. Staining of adjacent 2-microns sections for CD3, mast cell tryptase, and eosinophil cationic protein revealed that 90% of the IL-4 immunoreactive cells were mast cells, with biopsies from rhinitic subjects containing significantly more IL-4+ cells than biopsies from normal controls (p = 0.02), especially when assessed with the anti-IL-4 mAb 3H4. Mast cells also accounted for > 90% of IL-6 and > 50% of IL-5 immunoreactive cells. IL-5 immunoreactivity was also localized to eosinophils, whereas IL-8 localized predominantly to the nasal epithelium in both groups. No cytokines were found in association with T lymphocytes. These findings indicate that the mast cell is an important source of preformed cytokines and as such may contribute to the chronicity of the mucosal inflammation that characterizes allergic rhinitis.
IL-2 receptor-positive intrathyroidal lymphocytes in Graves' disease. Analysis of V alpha transcript microheterogeneity.
Authors: McIntosh R S , Tandon N , Pickerill A P , Davies R , Barnett D , Weetman A P
Published: December 2022

There has been considerable interest recently in the possible restriction of the TCR repertoire in autoimmune disorders, because such restriction would have important therapeutic implications. Reports of restriction of the TCR V alpha but not V beta repertoire in the thyroid in Graves' disease could not be repeated in an earlier study. Using RNA derived from matched peripheral blood, thyroid tissue, intrathyroidal lymphocytes (ITL), and IL-2R+ and IL-2R- subpopulations of ITL from Graves' patients, we conducted reverse transcription polymerase chain reaction/Southern blot analysis of TCR V alpha family usage. No evidence was found for V alpha restriction in the IL-2R+ subpopulation of ITL from eight patients, one of whom was operated on within 1 mo of diagnosis. We have further analyzed samples from seven of these patients by resolution on denaturing polyacrylamide gels. Typically, a single dominant band was amplified with surrounding minor bands in a normal distribution. Dominant and minor species were both the result of amplification from in frame V alpha transcripts, and the minor bands were separated in size by increments of 3 bp. We found no evidence for reduced heterogeneity of the V alpha transcripts in ITL or IL-2R+ ITL populations relative to peripheral blood in the vast majority of samples. This therefore suggests that there is little difference between the blood lymphocyte population and the activated T cell population in the thyroid in patients with Graves' disease, and indicates that in autoimmune thyroid disease, this method of analysis is not sufficient to distinguish between autoreactive and bystander T cell populations.
T cell-antigen-presenting cell interactions in human systemic lupus erythematosus. Evidence for heterogeneous expression of multiple defects.
Authors: Via C S , Tsokos G C , Bermas B , Clerici M , Shearer G M
Published: December 2022

To assess interactions between T cells and APC in patients with SLE, PBL were stimulated in vitro and IL-2 production measured after stimulation with either a MHC-self-restricted Ag (influenza A virus) or an Ag which can use both MHC-self-restricted or unrestricted T cell activation pathways (HLA-alloantigen). SLE patients (n = 26) and controls (n = 8) were categorized as responder (+) or nonresponder (-) for each stimulus and grouped according to their paired response pattern. All controls responded to both influenza virus (Flu) or alloantigen (Allo) and were categorized as +/+. In contrast, SLE patient response patterns were heterogeneous with no evidence for a single SLE-associated defect. Instead, SLE patient responses fell into one of three different patterns: a) normal responses to both Flu and Allo (+/+), observed in nine (35%) patients; b) defective responses to Flu but intact Allo responses (-/+) observed in 12 (46%) SLE patients; and c) defective responses to both Flu and Allo (-/-), observed in five (19%) SLE patients. There was no statistically significant correlation between immune response pattern and the use of immunosuppressants. Further analysis of -/- SLE patients indicated defects in APC function and in both CD4+ and CD8+ T cell function. In contrast, cell depletion and add-back studies in -/+ SLE patients demonstrated defects in APC function only. Thus, similar to the well recognized clinical heterogeneity among SLE patients, our data support the concept that SLE patients are heterogeneous with respect to in vitro T cell-APC function, exhibiting responses ranging from normal function to defects in APC and in both T cell subsets. Prospective studies are in progress to determine the clinical relevance of these observations.
A conservative mutation in a class I MHC molecule outside the peptide binding groove stimulates responses to self peptides.
Authors: Grandea A G , Bevan M J
Published: December 2022

A transgenic mouse has been made that expresses a mutant MHC class I H-2Kb molecule with glutamic acid at position 65 (E65) in place of glutamine. The side chain at position 65, on the outward face of the alpha-helix of the alpha 1 domain of the class I molecule, interacts with the TCR, and not with the peptide binding groove. The transgenic mouse, on a DBA/2 background, mounts Kb,E65-restricted Ag-specific responses to conventional Kb-restricted Ag such as OVA and vesicular stomatitis virus, and shows strong alloreactivity to wild-type Kb. The transgenic mouse also mounts a primary in vitro alloreactive response directed to a mutant molecule with aspartic acid at position 65 (D65). This response is relatively weak, probably because of the structural similarities between aspartic and glutamic acid side chains; both have carboxylic termini, and the aspartic acid side chain is shorter by a single secondary carbon. The alloreactive CTL lines elicited by this conservative change are cross-reactive among several position-65 variants of H-2Kb. Individual CTL clones are specific for self peptides that can be extracted from cells expressing Kb,E65, and from purified wild-type Kb molecules, and that are recognized in the context of the D65 residue. Thus, the smallest variance from self in a class I molecule, even outside the peptide binding groove, can be antigenic.
Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment.
Authors: Harding C V , Geuze H J
Published: December 2022

Exogenous protein Ag are processed within endocytic compartments to produce peptides that bind to class II MHC (MHC-II) molecules for presentation to T cells. We have now identified a subcellular compartment in which immunogenic peptides bind to MHC-II as a subset of high density lysosomes. Immunoelectron microscopy of whole cells and dense Percoll gradient subcellular fractions showed early tubulovesicular lysosomes with high levels of MHC-II. Typical mature lysosomes contained less MHC-II. Pulse-chase biosynthetic labeling of macrophages followed by immunoprecipitation of MHC-II from dense lysosomal fractions showed that MHC-II molecules targeted efficiently to lysosomes after biosynthesis. Moreover, lysosomal MHC-II molecules were rapidly loaded with immunogenic peptide (as detected by T cells) soon after exposure of macrophages to Ag and before similar expression of peptide-MHC-II complexes on the plasma membrane; this loading was blocked at 18 degrees C. We propose that nascent MHC-II molecules target to early tubulovesicular lysosomes and bind immunogenic peptides therein; the resulting peptide-MHC-II complexes are then transported to the plasma membrane.
Novel mechanism for inhibition of human T cells by glucocorticoids. Glucocorticoids inhibit signal transduction through IL-2 receptor.
Authors: Paliogianni F , Ahuja S S , Balow J P , Balow J E , Boumpas D T
Published: December 2022

Interaction of IL-2 with its high affinity membrane receptor complex (IL-2R) present on activated T lymphocytes induces cell proliferation and mediates effector functions. Glucocorticoids inhibit IL-2 production by inhibiting TCR-mediated signal transduction. We asked whether they also inhibit the action of IL-2 by inhibiting signal transduction through IL-2R. Human peripheral blood T cells, stimulated with PMA for 48 h (PMA blasts), were incubated with IL-2 in the presence of incremental dosages of dexamethasone (Dex; 10(-5)-10(-9) M). Dex inhibited the IL-2-dependent proliferation of PMA blasts in a dose-dependent fashion (IC50, 5 x 10(-8) M). Cell surface expression of IL-2R alpha- and beta-chains as determined by immunofluorescence analysis was not affected by Dex. In addition, Scatchard plot analysis of 125I-labeled IL-2 showed that Dex did not affect the binding of IL-2, thus suggesting that inhibition is due to a postreceptor effect. Inhibition of T cell proliferation by Dex was associated with decreased IL-2-dependent tyrosine phosphorylation of several intracellular proteins and decreased phosphorylation of the retinoblastoma gene product Rb, a protein essential for controlling the progression of cells through the cell cycle. IL-2-dependent IL-2R alpha expression in PMA blasts and NF-kB induction in resting human T cells were also inhibited by Dex. These results demonstrate that glucocorticoids inhibit preactivated T cells by down-regulating signal transduction through IL-2R.
Structural analysis of chicken factor B-like protease and comparison with mammalian complement proteins factor B and C2.
Authors: Kjalke M , Welinder K G , Koch C
Published: December 2022

Chicken complement factor B-like protease is a glycoprotein of 95 kDa. Activation of chicken serum complement with inulin cleaved the B-like protease into an N-terminal Ba fragment of 37 kDa and a C-terminal Bb fragment of 60 kDa. The whole protein and the two fragments were purified by affinity chromatography using mAb to chicken Ba or Bb followed by ion exchange chromatography. Amino acid sequencing showed that chicken B-like protease was cleaved at a site homologous to that cleaved in mammalian complement components B and C2 on activation. Limited tryptic digestion of the B-like protease generated fragments similar to Ba and Bb. More than 200 residues of the Ba sequence and two N-linked glycosylation sites were established by amino acid sequencing of peptides derived by digestion with four proteases. Comparison of human and mouse C2 and B sequences indicated a slower evolutionary rate for B (85% sequence identity) than for C2 (74% sequence identity). Comparison of chicken Ba to human and mouse C2b and Ba showed 42 to 45% sequence identity with respect to C2b fragments, and 46 to 49% sequence identity with respect to Ba fragments. Taking the slower evolutionary rate of factor B into account, chicken factor B-like protease seems to be equally related to mammalian complement components B and C2, and the B-like protease most likely represents the present-day descendant of a common ancestral protein for mammalian B and C2. This conclusion is in agreement with the requirement for the B-like protease in both classical and alternative activation pathways for chicken complement, and with the apparent lack of a chicken serum protein with exclusive C2 activity.
Lymphocyte-vascular adhesion protein-2 is a novel 70-kDa molecule involved in lymphocyte adhesion to vascular endothelium.
Authors: Airas L , Salmi M , Jalkanen S
Published: December 2022

Extravasation of leukocytes from the blood is essential for normal lymphocyte recirculation as well as in mounting an adequate inflammatory response in different tissues. Leukocyte migration from the blood is controlled by sophisticated interactions between surface receptors on leukocytes and their corresponding endothelial cell ligands. Here we describe a novel adhesion molecule, lymphocyte-vascular adhesion protein-2 (L-VAP-2), recognized by 4G4 mAb that was produced by immunizing mice with an enriched endothelial cell preparation isolated from inflamed human synovium. mAb 4G4 stains a subpopulation of venules in lymphoid and nonlymphoid tissues as well as a few high endothelial venules in lymphoid tissues. L-VAP-2 is constitutively expressed on human umbilical vein endothelial cells, and its expression cannot be up-regulated by cytokines and mitogens such as TNF-alpha, IL-1, and LPS. The Ag is expressed on approximately 20% of PBL, whereas granulocytes and monocytes are negative. On lymphocytes, L-VAP-2 is preferentially expressed on B cells and CD8+ T cells. The molecular mass of L-VAP-2 is approximately 70 kDa as determined from immunoprecipitated, 125I-labeled endothelial cell lysates. The involvement of L-VAP-2 in lymphocyte binding to endothelium was tested in vitro using human umbilical vein endothelial cells. Both the intact antibody and F(ab')2 fragments of it consistently inhibited lymphocyte binding to human umbilical vein endothelial cells by approximately 25%. On the basis of the molecular mass estimation and the staining of tissue sections, leukocyte populations, and ICAM-1 and ICAM-2 transfectants, L-VAP-2 appears to be a novel Ag involved in the lymphocyte-endothelial cell interaction.
Antagonistic effects of lipopolysaccharide binding protein and bactericidal/permeability-increasing protein on lipopolysaccharide-induced cytokine release by mononuclear phagocytes. Competition for binding to lipopolysaccharide.
Authors: Dentener M A , Von Asmuth E J , Francot G J , Marra M N , Buurman W A
Published: December 2022

Serum proteins play an important role in LPS-induced cell activation. The LPS binding protein (LBP) enhances cellular responses to LPS, whereas the polymorphonuclear leukocyte product bactericidal/permeability-increasing protein (BPI) inhibits LPS-induced cell activation. In this study the influences of LBP and BPI, two proteins with opposite effects, but with considerable sequence homology, on LPS-induced mononuclear phagocytic cell cytokine release was studied. LBP was shown to enhance LPS-induced TNF-alpha, IL-6, and IL-8 release by mononuclear phagocytic cells, whereas BPI inhibited the release of these cytokines. Furthermore, the effects of LBP and BPI on LPS-induced cytokine release by mononuclear phagocytic cells were shown to be counteractive. BPI interfered with the enhancing effect of LBP on the LPS-induced cytokine release. At high LBP to BPI ratios, BPI could no longer inhibit LBP-induced enhancement. In accordance, increasing concentrations of BPI abrogated the LBP effect. Next, it was shown that LBP and BPI compete for binding to LPS by using an assay system that detects binding of free BPI to an anti-BPI mAb. LPS prevented binding of BPI to anti-BPI mAb, whereas preincubation of LPS with LBP prevented the LPS-induced inhibition. Also, it was observed that both BPI and LBP inhibited LPS activity in the chromogenic LAL assay. We conclude from this study that LBP and BPI have counteractive effects on LPS-induced mononuclear phagocytic cell cytokine release by competing for binding to LPS.