Differential expression of cytokine genes in CD27-positive and -negative CD4 lymphocyte subsets from healthy humans and rheumatoid arthritis patients

Immunofluorescence analysis of CD27 expression by CD4 lymphocytes from the peripheral blood of healthy humans or rheumatoid arthritis (RA) patients and from the synovial fluid (SF) of RA patients was carried out, along with the estimation of cytokine gene [interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10 and interferon-gamma (IFN-gamma)] expression in these lymphocyte subsets by RT-PCR. Although no differences in CD27-positive and -negative peripheral blood CD4 cell subset distribution were revealed, marked differences in IL-3, IL-4, IL-5 and IFN-gamma mRNA expression were detected between these lymphocyte subsets and between control and disease states. These results showed that phenotyping of different cell subsets in disease cannot provide adequate information about lymphocyte functional status. To estimate differences in cytokine gene expression, CD4 lymphocytes from the peripheral blood and SF of RA patients were compared. In both cases, mRNAs for IL-2, IL-4, IL-10 and IFN-gamma were detected, but CD4 cells from SF failed to express detectable levels of IL-5 mRNA despite our findings of a CD27-cell accumulation within the synovial population of CD4 lymphocytes. These are the first data to demonstrate that expression of the IL-5 gene in RA SF CD27- lymphocytes is down-regulated and that IL-5 disregulation in RA cannot be ruled out.     

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.     

Identification of functional human splenic memory B cells by expression of CD148 and CD27

Memory B cells isolated from human tonsils are characterized by an activated cell surface phenotype, localization to mucosal epithelium, expression of somatically mutated immunoglobulin (Ig) variable (V) region genes, and a preferential differentiation into plasma cells in vitro. In spleens of both humans and rodents, a subset of memory B cells is believed to reside in the marginal zone of the white pulp. Similar to tonsil-derived memory B cells, splenic marginal zone B cells can be distinguished from naive follicular B cells by a distinct cell surface phenotype and by the presence of somatic mutations in their Ig V region genes. Although differences exist between human naive and memory B cells, no cell surface molecules have been identified that positively identify all memory B cells. In this study, we have examined the expression of the receptor-type protein tyrosine phosphatase CD148 on human B cells. CD148(+) B cells present in human spleen exhibited characteristics typical of memory B cells. These included an activated phenotype, localization to the marginal zone, the expression of somatically mutated Ig V region genes, and the preferential differentiation into plasma cells. In contrast, CD148(-) B cells appeared to be naive B cells due to localization to the mantle zone, the expression of surface antigens typical of unstimulated B cells, and the expression of unmutated Ig V region genes. Interestingly, CD148(+) B cells also coexpressed CD27, whereas CD148(-) B cells were CD27(-). These results identify CD148 and CD27 as markers which positively identify memory B cells present in human spleen. Thus, assessing expression of these molecules may be a convenient way to monitor the development of memory B cell responses in immunocompromised individuals or in vaccine trials.     

Expansion of mature thymocyte subsets before emigration to the periphery

A small population of DNA-synthesizing mature thymocytes could be defined by analyzing cell surface markers and 5-bromo-2'-deoxyuridine (BrdUrd) labeling by four-color cytofluorometry. These cells have a completely mature phenotype (CD4- CD8+ or CD4+ CD8- TCR(high), HSA-, Qa-2(high)) and expand only weakly after BrdUrd incorporation. They recovered immediately in total number and in DNA synthesis rate after treatment with the antimitotic drug demecolcin, thus much faster than immature CD4+ CD8+ thymocytes. These data demonstrate the existence of a late intrathymic expansion phase, independent of that of developing CD4+ CD8+ immature cells, and involving phenotypically mature cells renewed each day. In mixed chimeras prepared by transfer of bone marrow and lymph node cells into RAG-2(-/-) mice, all cycling mature thymocytes were bone marrow derived. They are thus produced in situ and do not correspond to peripheral T cells reentering the thymus. Double FITC/BrdUrd detection showed that a high proportion (10-20%) of recent thymic emigrants were BrdUrd+ just postcycling cells and that around 50% of cycling mature thymocytes are just ready to emigrate to the periphery in the few hours after DNA synthesis. The late intrathymic expansion phase demonstrated here increases the daily thymic cell export by at least 30%. It could play a role in the adjustment of the T cell repertoire before emigration and in the regulation of the thymic cell output into the peripheral T cell pool.     

Regulation of CD11b/CD18 expression in human neutrophils by phospholipase A2

Recent evidence suggests that phospholipase A2 (PLA2)-derived lipid mediators may regulate a number of neutrophil responses including degranulation and adhesion. In view of the potential role of PLA2 in stimulus-secretion coupling, we examined the relationship between PLA2 activation and the surface expression of CD11b/CD18 (MAC-1) in human polymorphonuclear leukocytes (hPMNL), including the functional consequences of PLA2 inactivation on MAC-1-dependent adhesion. The selective inhibition of PLA2 by the marine natural products manoalide (MLD) and scalaradial (SLD) blocks [3H]arachidonic acid (AA) release in calcium ionophore A23187-stimulated neutrophils, and also inhibits secretion of specific and azurophilic granule constituents. Additional studies demonstrate that MLD, SLD, and other less potent PLA2 inhibitors such as 4-bromophenacylbromide and nordihydroguiaretic acid inhibit the surface expression of MAC-1 (IC50: MLD, 0.33 microM; SLD, 0.23 microM; 4-bromophenacylbromide, 2.8 microM; NDGA, 3.5 microM) at concentrations similar to those at which they inhibit [3H]AA release. Inhibitors of cyclooxygenase, 5-lipoxygenase, protein kinase C, or calcium channel antagonists have no effect on MAC-1 expression. PLA2 inactivation also prevents MAC-1 up-regulation in hPMNL stimulated with FMLP, IL-8, TNF-alpha, PMA, or platelet activating factor. In FMLP-stimulated hPMNL, under conditions in which no secondary granule constituents are secreted, MAC-1 and alkaline phosphatase up-regulation from intracellular granules is inhibited by MLD and SLD. Functional assays also demonstrate that MLD and SLD block MAC-1-dependent adhesion of activated neutrophils to keyhole limpet hemocyanin at concentrations that block the surface expression of MAC-1. [3H]AA release and MAC-1 expression in MLD and SLD-treated hPMNL could be recovered in the presence of 1 mM hydroxylamine in a time-dependent fashion, consistent with reported data that MLD and SLD inactivate PLA2 through Schiff base formation. In summary, these data emphasize the role of PLA2 as a key regulator of MAC-1 expression in models of neutrophil adhesion.     

T-cell receptor BJ gene segment expression in human umbilical cord blood CD4+ and CD8+ T-cell subsets

By employing RT-PCR-based technology, followed by Southern-blot analysis, patterns of relative TRC BJ gene segment usage in human CD4+ and CD8+ umbilical cord blood T cells (UCT) from ten children were determined in relation to seven recombined TCR BV gene (sub) families (BV 3, 5S1, 6S1-3, 8, 9, 12 and 18). Normal frequency of usage of individual BJ members was observed to be extremely nonrandom. BJ usage in association with each BV was ranked and mean ranking values were calculated for individual BJs. Moreover, BJ family usage and family ranges as well as individual BJ over-representations were determined. In all these aspects of BJ exon expression, CD4+ and CD8+ UCT displayed similar distribution patterns. Comparisons of BJ usage in UCT subpopulations and in the adult peripheral blood lymphocyte (PBL) counterparts were performed and many similarities were observed. However, discrepancies in two parameters were recorded; contrary to observations in PBL, individual BJ over-representations were virtually absent in UCT, and significantly less wide BJ family ranges were demonstrated in CD8+ UCT relative to CD8+ PBL T cells. These differences support the notion that UCT are in a less dynamic state than are PBL T cells. Hence, despite the fact that PBL T cells are subjected to continuous antigenic challenge, the striking resemblance of PBL and UCT with regard to the overall individual relative usage, ranking, mean ranking and family utilisation of BJ gene segments, irrespective of the choice of recombined BV exons, may suggest a relatively nondiscriminatory role for the BJ gene product in antigen recognition as compared to those encoded by the BV, (N) and BD gene segments.     

Differential expression of CD40 ligand on T cell subsets. Implications for different roles of CD45RA+ and CD45RO+ cells in IgE production

Physical contact between human T lymphocytes and B lymphocytes is required for the induction of IgE production. In the present study, we examined the abilities of CD45RA+ and CD45RO+ human T cell subsets to provide help for IgE production by human peripheral blood B cells in the presence of IL-4. Purified peripheral CD45RA+ T cells are much better inducers of IgE synthesis than are CD45RO+ T cells. Activation of CD45RA+ T cells, but not CD45RO+ T cells, via the TCR/CD3 complex is sufficient to confer the ability to provide IgE help, suggesting that an inducible T cell surface molecule plays an important role in this system. The CD40 ligand, an inducible T cell surface molecule, is expressed at higher levels on CD45RA+ T cells as compared with CD45RO+ T cells following CD3-stimulation. Blocking of the CD40-CD40 ligand interaction in vitro by the addition of a soluble form of B cell CD40 Ag completely blocks IgE production induced by CD45RA+ T cells. Finally, the in vitro conversion of CD45RA+ T cells to the CD45RO+ phenotype is accompanied by a loss in the ability of these cells to express the CD40 ligand in response to anti-CD3 stimulation as well as a loss in their ability to provide IgE help. These results suggest that both CD45 subsets may play significant and distinct roles in the induction of IgE production under physiologic conditions: CD45RO+ T cells provide IL-4 and the CD45RA+ subset provides the second signal via the CD40 ligand.     

RANTES chemokine expression is related to acute cardiac cellular rejection and infiltration by CD45RO T-lymphocytes and macrophages

BACKGROUND: Despite the advances made in immunosuppression therapy, episodes of acute cellular rejection may affect graft function and survival. We investigated the role of RANTES in cellular recruitment and in cardiac allograft rejection. METHODS: Endomyocardial biopsies (n = 65) from 30 patients were taken at various times after transplantation. In 4 subjects who died of acute cellular rejection, the profile of RANTES expression was monitored in all biopsy specimens and in postmortem tissue. Myocardial tissue from 10 other transplants was also analyzed. Sections were stained with an anti-human RANTES antibody with the streptavidin-biotin technique. RANTES-positive cells were related to macrophage, CD45RO "memory" T-cell, and eosinophil infiltration. RESULTS: RANTES-positive cells were identified within the cellular infiltrate in 95% of biopsies with moderate/severe rejection and 28% with mild rejection. RANTES-positive, CD45RO-positive, and macrophage cell numbers were higher in subjects who died of acute cellular rejection than of other causes. A highly significant difference in RANTES-positive cell number was observed between moderate/severe, mild, and nonrejection groups (p = .0001) and correlated significantly with macrophage number in both right and left ventricles (r = .693, p < .01; r = .599, p < .05, respectively) and with the number of "memory" T cells (r = .829, p < .001; r = .779, p < .01, respectively). CONCLUSIONS: These findings suggest that local release of RANTES is important in the recruitment of both macrophages and CD45RO T cells in cardiac allograft rejection. RANTES may be an important chemokine to target for therapeutic intervention in heart rejection.     

Regulation of interleukin-13 receptor constituents on mature human B lymphocytes

Human B cells stimulated through both their immunoglobulin and CD40 receptors up-regulate 745 +/- 51 interleukin (IL)-13 ligand binding sites with an affinity of 0.91 +/- 0.08 nM within 24 h. IL-13 binds primarily to the IL-13Ralpha1 with subsequent sequestration of the IL-4Ralpha into the complex. IL-13Ralpha1 may also be found in those receptors capable of binding IL-4. gamma chain (gammac) participates in receptors capable of binding IL-4 but is not found in association with bound IL-13. Dimeric receptors composed of the IL-4Ralpha complexed with either the IL-13Ralpha1 or gammac occur simultaneously within defined B cell populations. mRNAs for all receptor constituents are increased subsequent to immunoglobulin stimulation alone, while maximal expression of IL-13Ralpha1 is more dependent upon co-stimulation of immunoglobulin and CD40 receptors. mRNA levels for IL-13Ralpha1 vary over a wider range subsequent to surface stimulation than other receptor components. Although gammac is not bound to IL-13 in B cells under the conditions evaluated, it may influence IL-13 binding by competing with IL-13Ralpha1 for association/sequestration with the IL-4Ralpha chain. IL-13Ralpha2 does not participate in the IL-13 receptor that is up-regulated upon activation of quiescent tonsillar B lymphocytes, although mRNA for the protein may be found in the centroblastic fraction of tonsillar cells.     

Regulation of heat shock protein 27 expression of prostatic cells in response to heat treatment

BACKGROUND: Reading acuity as well as reading speed are good predictors of everyday visual function. As visual acuity tests are poor predictors of the real-world function, performance-based tests, e.g., reading speed measurements, can be used for the determination of visual function. Thus, a German reading chart was developed in order to evaluate reading acuity as well as reading speed. METHODS: Print size is defined as the height of a lower case x and progresses logarithmically from one phrase to another (factor: 1.25). Reading acuity is determined in LogRAD (Reading Acuity Determination). 32 short German phrases were created, comparable concerning grammatical difficulty as well as in number (n = 14), length and position of words. The reading speed parameters measured with a stop-watch in 160 persons (aged: Phi = 21a +/- 3.8a) were calculated in words per minute (w/min). Out of the 32 phrases the 24 most similar ones were selected statistically and used for the reading charts (Radner Reading Charts). With these reading charts a reading acuity score (LogRAD-score) can be calculated considering reading errors in words of different length. Reading speed can be determined at the same time. Reading acuity (LogRAD-Score) was measured in 32 normal eyes of 16 students and compared to the angular visual acuity (LogMAR). RESULTS: The mean reading speed of the test persons was 211.8 +/- 34.1 w/min. 24 phrases fulfilled the test item criteria for the reading chart: mean +/- 0.25 x SD. The reliability analyses yielded an overall Cronbach's alpha coefficient of 0.98! The mean visual acuity measured in 32 eyes was -0.115 +/- 0.097 LogMAR and the mean reading acuity score was +0.026 +/- 0.091 LogRAD. The mean difference was +0.104 +/- 0.066 and the correlation between LogMAR and LogRAD was good (r = 0.59). CONCLUSIONS: With these reading charts it is for the first time possible to simultaneously determine reading acuity as well as reading speed in German. The high reliability of the 24 phrases and the high correlation between LogMAR and LogRAD leads us to expect a good reproducibility of the reading acuity evaluations. For the "Radner Reading Charts" we have shown that print size is the main reason for changes of reading speed.     

Enhancement of antitumor immunity by expression of CD70 (CD27 ligand) or CD154 (CD40 ligand) costimulatory molecules in tumor cells

BACKGROUND: Most catalytic RNAs depend on divalent metal ions for folding and catalysis. A thorough structure-function analysis of catalytic RNA therefore requires the identification of the metal-ion-binding sites. Here, we probed the binding sites using Fenton chemistry, which makes use of the ability of Fe2+ to functionally or structurally replace Mg2+ at ion-binding sites and to generate short-lived and highly reactive hydroxyl radicals that can cleave nucleic acid and protein backbones in spatial proximity of these ion-binding sites. RESULTS: Incubation of group I intron RNA with Fe2+, sodium ascorbate and hydrogen peroxide yields distinctly cleaved regions that occur only in the correctly folded RNA in the presence of Mg2+ and can be competed by additional Mg2+, suggesting that Fe2+ and Mg2+ interact with the same sites. Cleaved regions in the catalytic core are conserved for three different group I introns, and there is good correlation between metal-ion-binding sites determined using our method and those determined using other techniques. In a model of the T4 phage-derived td intron, cleaved regions separated in the secondary structure come together in three-dimensional space to form several metal-ion-binding pockets. CONCLUSIONS: In contrast to structural probing with Fe2+/EDTA, cleavage with Fe2+ detects metal-ion-binding sites located primarily in the inside of the RNA. Essentially all metal-ion-binding pockets detected are formed by tertiary structure elements. Using this method, we confirmed proposed metal-ion-binding sites and identified new ones in group I intron RNAs. This approach should allow the localization of metal-ion-binding sites in RNAs of interest.     

An enhancer that directs lineage-specific expression of CD8 in positively selected thymocytes and mature T cells

Positive selection of CD4+CD8+ T cells to the CD4+CD8- helper and CD4- CD8+ cytotoxic lineages is a multistep process that involves complex regulation of coreceptor gene expression. By analyzing expression of a reporter gene in transgenic mice, we have identified a DNA segment, located between the murine CD8beta and CD8alpha genes, that has enhancer activity restricted to CD8 lineage cells. Remarkably, this enhancer functions in thymocytes undergoing positive selection to the CD4-CD8+ phenotype but not in immature double-positive thymocytes. The enhancer also functions in gut intraepithelial lymphocytes that express CD8alpha but not CD8beta, suggesting that it is specific for CD8alpha expression. The tight correlation between activation of this enhancer and the final step in positive selection has important implications for understanding the mechanism of lineage commitment in thymocytes.     

Soluble CD27 in thyroid disorders

Central acinic cell carcinoma is extremely rare; only six cases have been reported in the literature. An unusual case of central acinic cell carcinoma of the mandible in an 84-year-old Japanese woman is presented. This is thought to be the seventh case of central acinic cell carcinoma described in the English literature.     

Functional CD4+ T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependent epitope of CD45 molecules (JH6.2) and one a natural thymocytotoxic autoantibody (NTA) with an unknown reactive epitope (NTA260), we subdivided splenic CD4+ T cells from 2-month-old BALB/c mice into five phenotypically distinct subsets. CD45RC+NTA260- (S I) cells were phenotypically analogous to CD4+ T cells predominating in newborn mice and produced a significant amount of IL-2, but not so IL-4, IL-10 or IFN-gamma when stimulated with immobilized anti-CD3 mAb in vitro. They appeared to consist mainly of naive ThP cells. The CD45RC+NTA260+ (S II) subset also produced IL-2, but not other cytokines; however, the IL-2 levels produced were much higher than seen with the S I subset, thereby suggesting the predominance of further maturated ThP cells. The CD45RC-NTA260+ (S III) subset mainly produced IL-4, IL-10, IFN-gamma and less IL-2, and contained memory cells that helped the secondary antibody response to a recall antigen, and hence contained Th2 and probably a mixture of Th0 and Th1 cells. The CD45RC-NTA260- (S IV) subset was a poor responder to the immobilized anti-CD3 mAb. The CD45RCbrightNTA260dull (S V) subset consisted of a small number of cells that were phenotypically analogous to activated CD4+ T cells. While an age-associated decrease in the proportion of S I and less markedly in S II and in turn increase in S III subsets of CD4+ T cells occurred in normal BALB/c mice, autoimmune disease-prone (NZB x NZW)F1 mice showed a marked age-associated decrease in the proportion of not only S I, II but also III subsets. As aged (NZB x NZW)F1 mice carry CD4+ T helper cells for IgG anti-DNA antibody production, such age-associated polarization to the S IV subset appears to be critical in the pathogenesis of autoimmune disease in these mice.     

T cell responses to heat-shock protein 60: differential responses by CD4+ T cell subsets according to their expression of CD45 isotypes

We demonstrate that human T lymphocytes proliferate in vitro to highly purified human heat-shock protein 60 (Hu.hsp60). The response to this self Ag was confined to the CD45RA+ RO- T cell subset, with minimal responses by adult CD45RA- RO+ T cells. Experiments using keyhole limpet hemocyanin as a prototypic novel Ag, or tetanus toxoid as a recall Ag, were consistent with the notion that CD45RA+ RO- and CD45RA- RO+ T cell subsets can be designated as naive and memory cells, respectively; thus, responses to Hu.hsp60 were confined to the putative naive subset. In contrast, both CD45RA+ RO- and CD45RA- RO+ T cell populations proliferated to bacterial hsp60 from Mycobacterium leprae, Escherichia coli, or Chlamydia trachomatis. However, only CD45RA- RO+ (memory) T cells responded to a mycobacterial hsp60-derived peptide previously defined as a major bacteria-specific epitope. Experiments with cord blood T cells, which are CD45RA+ RO- and can be considered truly naive, showed that the peptide could elicit responses from naive T cells in vitro; cord blood cells also responded to Hu.hsp60. Since bacterial hsp60 Ags contain both conserved and nonconserved epitopes, we speculate that in vivo challenge with bacterial hsp60 will activate T cells capable of seeing either type of epitope, but only those that see nonconserved epitopes maintain the CD45RA- RO+ memory phenotype. However, T cells recognizing conserved epitopes, while not apparently being recruited to the memory pool, may nevertheless play a role in immunoregulation, particularly in the context of inflammation, when expression of Hu.hsp60 is increased.     

Acetylcholine synthesis and muscarinic receptor subtype mRNA expression in T-lymphocytes

We used a sensitive and specific radioimmunoassay for acetylcholine (ACh), and detected significant amounts of ACh in the blood of various mammals, including humans. About 60% of human blood ACh was localized in mononuclear leukocytes. Human leukemic T-cell lines, used as T-lymphocyte models, contained both ACh and choline acetyltransferase (ChAT) activity. Furthermore, ChAT mRNA and protein were detected in the T-cell line MOLT-3. Phytohemagglutinin, a T-cell activator, increased both synthesis and release of ACh by MOLT-3 cells. Muscarinic receptor subtype mRNA expression was confirmed in various T-cell lines. These findings indicate that ACh synthesized by ChAT in T-lymphocytes acts on the muscarinic receptors on lymphocytes in autocrine and/or paracrine pathways and suggest that ACh in blood functions as a modulator of T-cell-dependent immune responses.     

Synergistic augmentative effect of interleukin-10 and CD27/CD70 interactions on B-cell immunoglobulin synthesis

BACKGROUND: Patients who complain of constipation can be divided into those who have lost the natural call to stool, but develop abdominal discomfort after several days without a bowel movement (no urge); and those who experience a constant sensation of incomplete evacuation (urge). AIMS: To determine whether the two groups differ in symptoms, colonic transit, and perceptual responses to controlled rectal distension. METHODS: Forty four patients with constipation were evaluated with a bowel symptom questionnaire, colonic transit (radiopaque markers), and rectal balloon distension. Stool (S) and discomfort (D) thresholds to slow ramp (40 ml/min) and rapid phasic distension (870 ml/min) were determined with an electronic distension device. Fifteen healthy controls were also studied. RESULTS: All patients had Rome positive irritable bowel syndrome (IBS); 17 were no urge and 27 urge. Mean D threshold to phasic rectal distensions was 28 (3) mm Hg in no urge, 27 (3) mm Hg in urge (NS), but higher in the control group (46 (2) mm Hg; p < 0.01). Sixty seven per cent of no urge and 69% of urge were hypersensitive for D. Slow ramp distension thresholds were higher in no urge (S: 26 (3); D: 45 (4) mm Hg) compared with urge (S: 16 (2); D: 31 (3) mm Hg; p < 0.01), or with controls (S: 15 (1); D: 30 (3); p < 0.01). CONCLUSIONS: Hyposensitivity to slow rectal distension is found in patients with IBS who complain of constipation and have lost the call to stool even though their sensitivity to phasic distension is increased.