Mitogen-activated Peripheral Blood Mononuclear Cells Research Articles

Constitutive Activation of STATs Upon In Vivo Human Immunodeficiency Virus Infection

Infection by the human immunodeficiency virus (HIV) either upregulates or downregulates the expression of several cytokines and interferons (IFNs) that use the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway for signal transduction. However, very little is known on the state of activation of the JAK/STAT pathway after HIV infection either in vivo or in vitro. In this regard, we report here that a constitutive activation of a C-terminal truncated STAT5 (STAT5▵) and of STAT1 occurs in the majority (∼75%) of individuals with progressive HIV disease. We have further demonstrated that, among peripheral blood mononuclear cells (PBMCs), STAT5▵ is activated preferentially in CD4+ T cells. In contrast to a published report, expression of STATs from PBMCs of infected individuals was comparable with that of seronegative donors. In addition, in vitro infection of mitogen-activated PBMCs with a panel of laboratory-adapted and primary HIV strains characterized by differential usage of chemokine coreceptors did not affect STAT protein levels. However, enhanced activation of STAT was observed after in vitro infection of resting PBMCs and nonadherent PBMCs by different viral strains. Thus, constitutive STAT activation in CD4+T lymphocytes represents a novel finding of interest also as a potential new marker of immunological reconstitution of HIV-infected individuals.

A cell line-based neutralization assay for primary human immunodeficiency virus type 1 isolates that use either the CCR5 or the CXCR4 coreceptor.

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.

Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs.

Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.

Down-modulation of the ZAP-70 protein tyrosine kinase in macaque T lymphocytes infected with SIVsmmPBj14.

The simian immunodeficiency virus SIV-PBj14 is the most virulent primate lentivirus identified to date. Other SIV strains, including the parental SIVsmm9, require mitogen-activated peripheral blood mononuclear cells (PBMC) for replication in vitro; however, SIV-PBj14 replicates in quiescent pig-tailed macaque PBMC and induces cellular proliferation, consistent with its in vivo pathogenesis. To identify mechanisms involved in SIV-PBj14-induced T-cell proliferation, kinases important in early T-cell receptor-mediated activation pathways were studied. Immunoblot analyses showed that ZAP-70 protein, a tyrosine kinase, was downregulated, primarily in CD8+ T cells, as early as 30 minutes after in vitro infection of quiescent macaque PBMC with SIV-PBj 14. Furthermore, this downregulation required the presence of either CD4+ T cells or adherent cells or both cell populations. In agreement with the in vitro results, ZAP-70 expression was downregulated in macaque PBMC, spleen, and rectal lymph node cells as early as 2 days after rectal inoculation of pig-tailed macaques with SIV-PBj14. This phenomenon, however, was not observed in cells obtained from distal lymph nodes to which the virus had not disseminated, implying that the presence of SIV-PBj14 is necessary to induce downregulation of ZAP-70.

Reduced interferon-γ but normal IL-4 and IL-5 release by peripheral blood mononuclear cells from Xhosa children with atopic asthma

Background: Allergic asthma is increasing in black South Africans, a cohort with inherently high basal IgE levels. Atopy has been linked to an excess of the T helper 2 cytokines IL-4 and IL-5 relative to the T helper 1 cytokine interferon-γ (IFN-γ); however, most studies have utilized T cell clones. Studies on peripheral blood mononuclear cells (PBMC) have shown decreased IFN-γ release in patients with atopic dermatitis. It is uncertain whether this finding extends to atopic asthma. Objectives: To characterize cytokine release by mitogen-activated PBMC from Xhosa children and to investigate whether reduced IFN-γ release is a feature of atopic asthma and whether there is a relationship between cytokine profiles and asthma severity. Methods: Cytokine release and proliferation of phytohemagglutinin-stimulated PBMC from 10 patients with severe asthma and 14 patients with moderate asthma (highly allergic to house dust mites) and 17 healthy controls was assessed. Total serum, allergen-specific, and Ascaris-specific IgE was measured. Results: Proliferation did not differ between the groups. The release of IFN-γ was progressively decreased (and the IL-4/IFN-γ ratio increased) in the groups with moderate or severe asthma. Tumor necrosis factor-α release was reduced, but IL-4, IL-5, and granulocyte-macrophage–colony stimulating factor release was unchanged. The presence of Ascaris-specific IgE did not influence the cytokine profiles. Conclusion: Our study extends the findings observed for other atopic disorders and suggests that defective IFN-γ release is a generalized feature of atopic diseases. This study—the first to investigate both severe and moderate asthma, with the groups having similar atopic profiles—indicates that the extent of the defect in IFN-γ release might be related to asthma severity. ( J Allergy Clin Immunol 1997;100:662-8.)

Functional IL-2 receptors are expressed by rat lung type II epithelial cells.

Interferon (IFN)-gamma-induced inhibition of type II epithelial cell thymidine incorporation (45% decrease vs. control) was restored by cocultures with mitogen-activated peripheral blood mononuclear cells (PBMC) and conditioned media (CM) from mitogen-activated PBMC. Successive exposure of type II cells to IFN-gamma and interleukin (IL)-2 produced similar alterations in thymidine incorporation. Given that IL-2 is a powerful pleiotropic cytokine produced by lymphoid and myeloid cells, the presence of IL-2 receptors (IL-2R) was assessed in primary cultures of rat type II pneumocytes (TIIP) and the nontransformed alveolar type II epithelial cell line L2. The presence of IL-2R membrane protein on rat type II cells was established by immunodetection assays. The expression of all three murine IL-2R alpha-, beta-, and gamma-chain RNA transcripts in primary TIIP cultures and L2 cells was highlighted by reverse transcriptase-polymerase chain reaction analysis. Overall, these experiments demonstrate, for the first time, that type II epithelial cells can express functional IL-2R, confirming TIIP as a potential "partner" in the lung immune system. Consequently, it can be speculated that TIIP are new cellular targets for lymphokines using (IL-2R) gamma-chain-bearing receptors in lung distal air-spaces.

Resting Cell Neutralization Assay for HIV-1 Primary Isolates

A technique is described for detecting the activity of neutralizing polyclonal or monoclonal antibodies against HIV-1 primary isolates. Most commonly, neutralizing antibody activity for HIV-1 is assessed by quantifying the ability of antibodies to inhibit virus infection in mitogen-activated peripheral blood mononuclear cells or transformed lymphocytes. Because the target of HIV infectionin vivois neither a mitogen-activated nor a transformed cell, an assay using unstimulated peripheral blood mononuclear cells as a more physiologic target cell was developed. This “resting cell assay” mainly utilizes primary HIV-1 isolates that have been carried for only a few passagesin vitro.The result is an assay that is more efficient to perform and that detects neutralizing activity with comparable or greater sensitivity than that previously described for assays of primary HIV-1 isolates.

Products of enteropathogenic E. coli inhibit lymphokine production by gastrointestinal lymphocytes.

Previously we have shown that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated peripheral blood mononuclear cells (PBMCs). The aim of the present study was to determine whether products of EPEC alter lymphokine expression by gastrointestinal mucosal lymphocytes. Lysates from EPEC clones inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, IL-5, and interferon-gamma (IFN-gamma) but not IL-8 mRNA expression by lamina propria mononuclear cells isolated from surgically resected colon specimens. Inhibitory lysates did not significantly change CD25 expression on either CD4, CD8, or CD45R0 lymphocytes by flow cytometry. Bacterial supernatants of EPEC inhibited IL-2 and IL-5 protein secretion by mitogen-stimulated PBMCs. EPEC lysates inhibited IL-2 mRNA expression induced by lysates of nonpathogenic E. coli. In conclusion, EPEC contains a novel gene(s) that encodes factors that selectively inhibit IL-2, IL-4, IL-5, and IFN-gamma expression by mucosal mononuclear cells without affecting CD25 or IL-8 expression. Thus enteric bacteria can produce factors that may regulate the function of the gastrointestinal mucosal immune system.

Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes

Certain anti-cancer agents are known to induce apoptosis in human tumour cells. However, these agents are intrinsically cytotoxic against cells of normal tissue origin, including myelocytes and immunocytes. Here we show that a naturally occurring flavone of citrus origin, tangeretin (5,6,7,8,4'-pentamethoxyflavone), induces apoptosis in human promyelocytic leukaemia HL-60 cells, whereas the flavone showed no cytotoxicity against human peripheral blood mononuclear cells (PBMCs). The growth of HL-60 cells in vitro assessed by [3H]thymidine incorporation or tetrazolium crystal formation was strongly suppressed in the presence of tangeretin; the IC50 values range between 0.062 and 0.173 microM. Apoptosis of HL-60 cells, assessed by cell morphology and DNA fragmentation, was demonstrated in the presence of > 2.7 microM tangeretin. Flow cytometric analysis of tangeretin-treated HL-60 cells also demonstrated apoptotic cells with low DNA content and showed a decrease of G1 cells and a concomitant increase of S and/or G2/M cells. Apoptosis was evident after 24 h of incubation with tangeretin, and the tangeretin effect as assessed by DNA fragmentation or growth inhibition was significantly attenuated in the presence of Zn2+, which is known to inhibit Ca(2+)-dependent endonuclease activity. Ca2+ and Mg2+, in contrast, promoted the effect of tangeretin. Cycloheximide significantly decreased the tangeretin effect on HL-60 cell growth, suggesting that protein synthesis is required for flavonoid-induced apoptosis. Tangeretin showed no cytotoxicity against either HL-60 cells or mitogen-activated PBMCs even at high concentration (27 microM) as determined by a dye exclusion test. Moreover, the flavonoid was less effective on growth of human T-lymphocytic leukaemia MOLT-4 cells or on blastogenesis of PBMCs. These results suggest that tangeretin inhibits growth of HL-60 cells in vitro, partially through induction of apoptosis, without causing serious side-effects on immune cells.

Serum neutralization of feline immunodeficiency virus is markedly dependent on passage history of the virus and host system

Sera from feline immunodeficiency virus (FIV)-infected cats exhibited extremely low levels of neutralizing antibodies against virus passaged a few times in vitro (low passage), when residual infectivity was assayed in the CD3+ CD4- CD8- MBM lymphoid cell line or mitogen-activated peripheral blood mononuclear cells. By sharp contrast, elevated titers of highly efficient neutralizing activity against FIV were measured, by use of high-passage virus, in assays on either the fibroblastoid CrFK or MBM cell line. However, high-passage virus behaved the same as low-passage virus after one in vivo passage in a specific-pathogen-free cat and reisolation. Subneutralizing concentrations of infected cat sera enhanced the production of low-passage virus by MBM cells, an effect not seen with high-passage virus in CrFK cells. These qualitative and quantitative discrepancies could not be attributed to differences in the amount of immunoreactive viral material, to the amount of infectious virus present in the viral stocks, or to the presence of anti-cell antibodies. The observed effects were most likely due to the different passage history of the viral preparations used. The observation that neutralizing antibodies detected with high-passage virus were broadly cross-reactive in assays with CrFK cells but isolate specific in MBM cells suggests also that the cell substrate can influence the result of FIV neutralization assays. This possibility could not be tested directly because FIV adapted to grow in CrFK cells had little infectivity for lymphoid cells and vice versa. In vitro exposure to infected cat sera had little or no effect on the ability of in vivo-passaged FIV to infect cats. These data reveal no obvious relationship between titers against high-passage virus and ability to block infectivity of FIV in cats and suggest caution in the use of such assays to measure vaccine efficacy. In conclusion, by contrast with what has been previously reported for the use of CrFK cells and high-passage virus, both natural and experimental infections of cats with FIV generate poor neutralizing antibody responses with regard to in vivo protection.

The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.

Considerable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-1 nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef+ HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nef mutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef+ and Nef- viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef- viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact nef for subsequent viral growth. We now provide evidence that the Nef+ HIV is more infectious than Nef- HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five- to sixfold more infected cells if Nef+ viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef+ virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef+ and Nef- viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef+ infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMC-derived Nef+ HIC.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation, characterization, and transmission of human T-lymphotropic virus types I and II in culture.

Highly sensitive coculture methods were developed both for isolation of human T-lymphotropic virus types I and II (HTLV-1 and HTLV-II) from infected individuals and for productive infection of lymphoid cells. Mitogen-activated peripheral blood mononuclear cells (PBMC) from 13 HTLV-I- and 20 HTLV-II-positive specimens were cocultured with an equal number of mitogen-activated PBMC from HTLV-seronegative individuals, and culture supernatants were tested for the presence of soluble p24gag antigens at weekly intervals for 4 weeks. Eleven of 13 (85%) HTLV-I and 14 of 20 (70%) HTLV-II cultures were positive for p24 antigens. None of the 17 HTLV-seroindeterminate or six HTLV-seronegative specimens were positive for the presence of p24 antigen. The isolation rates for HTLV-I and HTLV-II by an alternative whole-blood lysis procedure were comparable to those obtained by standard PBMC cultures. Furthermore, cocultivation of PHA-stimulated PBMC from healthy donors with lethally irradiated HTLV-I- and HTLV-II-infected cell lines (SP and Mo-T, respectively) resulted in productive viral infection, as reflected by the appearance of p24gag antigens concomitant with specific genomic amplification of HTLV proviral DNA after 3 weeks of cocultivation. Thus, the cocultivation technique provides a highly sensitive and specific procedure both for HTLV isolation and for infection of target cells.

Binding of phycoerythrin-conjugated interleukin-6 to in vitro-activated human peripheral blood mononuclear cells--effect of immunosuppressive agents and of a calcium channel blocker.

We studied the effect of cyclosporine A, prednisolone, and the Ca2+ channel blocker verapamil on interleukin-6 binding to mitogen-activated peripheral blood mononuclear cells, using a flow cytometric technique and phycoerythrin-conjugated IL-6. All mitogenic stimuli up-regulated IL-6 binding to a variable degree. PHA alone or in combination with PMA was the most effective stimulant in up-regulating IL-6 binding in all the experiments performed. The main changes in IL-6 binding were seen in the large cell cluster, which consisted mainly of lymphoblasts. PHA and PHA/PMA, however, also up-regulated the mean fluorescence intensity on the small cell cluster, which consisted mainly of quiescent lymphocytes. The overall effect of the three pharmacological agents on mitogen-up-regulated IL-6 binding was minimal; most significant were a down-regulation by all three agents of IL-6 binding by small lymphocytes in PHA/PMA cultures, a down-regulation of IL-6 binding by CsA in PHA/PMA-induced large PBMC, and an up-regulation by verapamil of PMA-induced IL-6 binding in large PBMC. Measurements of IL-2 binding and of IL-6 production in the same cultures showed a different pattern than that seen with IL-6 binding, as well as different CsA, prednisolone, and verapamil action. In conclusion, by using a new flow cytometric technique providing information both about the quantity of bound cytokine and about the proportion of IL-6-binding cells, we have demonstrated that IL-6 receptor expression in vitro by PBMC can be up-regulated by the use of stimulants differing in the signal transduction pathways they activate. In addition, by using different pharmacological agents and stimuli to dissect different activation pathways of the in vitro immune response, we conclude that IL-6R generation is regulated differently from IL-6 production. Furthermore, since CsA and prednisolone are known inhibitors of in vitro IL-2 production, our results indicate that IL-6R generation does not rely exclusively on the presence of IL-2.

Expression and Secretion of IL-2 Receptor in Trauma Patients

The high-affinity cell-surface receptor for interleukin-2 (IL-2R) consists of the 75-kilodalton (kd) chain and a 55-kd glycoprotein known as Tac. This report examines the cellular expression of IL-2R and secretion of the soluble form of Tac in immunosuppressed blunt trauma (n = 20, injury severity score -20) and thermally injured (n = 20, Total body surface area greater than 35%) patients. The percentage of IL-2R-expressing peripheral blood mononuclear cells (PBMC) in mitogen-stimulated cultures from patients and age-matched normal donors was determined by direct immunofluorescence with a monoclonal anti-Tac followed by flow cytometry analysis. Levels of soluble Tac in patient sera were measured by a monoclonal antibody-based enzyme-linked immunosorbent assay. Expression of Tac antigen by mitogen-activated PBMC cultures from blunt trauma patients was transiently reduced (by as much as 40%) in 4 of 14 patients examined. Levels of serum Tac were significantly elevated (p less than 0.05) in 15 of 20 blunt trauma patients ranging from 750 to 3000 U/mL compared with 180 to 420 U/mL in control subjects. All burn patients studied 10 to 50 days after the injury demonstrated a significant reduction (by 50% to more than 90%) in the percentage of IL-2 receptor-bearing cells. Similarly serum levels of IL-2R increased significantly (p less than 0.001 to 0.05) in all burn patients studied, reaching concentrations as high as 5500 U/mL. These results suggest that major trauma, whether mechanical or thermal, induces alterations in the T-lymphocyte activation process. However the degree and duration of such changes may vary based on the nature of the injury.

POSSIBLE ASSOCIATION OF THE IMMUNOSUPPRESSIVE AND B CELL LYMPHOMA-PROMOTING PROPERTIES OF CYCLOSPORINE

Central to the immunosuppressive properties of cyclosporine is a drug imposed blockade of the interleukin-2 gene activation. As IL-6 stimulates antigen-activated T cells to release IL-2, we examined the influence of CsA on IL-6 gene expression and IL-6-supported T cell proliferation. Northern blot analysis revealed that CsA failed to abolish IL-6 gene expression in mitogen-activated peripheral blood mononuclear cells. In fact, increased IL-6 gene transcription and increased release of IL-6 bioactivity were detected using mitogen-activated PBMCs cultured with CsA doses (200-800 ng/ml) only slightly in excess of the minimal antiproliferative dose. CsA completely abrogated the IL-6-stimulated proliferative responses of macrophage-depleted T cells stimulated with polyvalent anti-CD3 monoclonal antibodies. It is interesting that CsA-treated patients evidence an increased incidence of polyclonal lymphoproliferative disorders and B cell lymphomas. As IL-6 fosters B cell activation and growth of EBV-transformed B cells, excessive CsA doses may support development of EBV-transformed B cell lymphomas via superinduction of the IL-6 gene.

Feline Immunodeficiency Virus (FIV) Infection in the Cat as a Model for HIV Infection in Man: FIV-Induced Impairment of Immune Function

To assess the value of feline immunodeficiency virus (FIV) infection as a model for human immunodeficiency virus (HIV) infection in man, we studied the impairment of certain immunological functions following natural or experimental FIV infection. Proliferative responses of peripheral blood mononuclear cells (PBMC) from symptomatic and asymptomatic cats after naturally or experimentally acquired FIV infection, induced by activation with the mitogens concanavalin A, pokeweed mitogen, or lipopolysaccharide or by stimulation with human interleukin-2 (IL-2), were significantly lower than the proliferative responses found with PBMC from noninfected control cats. Also IL-2 production levels of mitogen-activated PBMC from naturally infected symptomatic cats were significantly reduced. These data confirm that the pathogenesis of FIV infection in the cat, like HIV infection in man, is characterized by a serious malfunction of the immune system.

Production of B‐cell growth factor interleukin 2 and gamma interferon by peripheral blood lymphocytes from patients with chronic lymphocytic leukemia of B‐cell type

Chronic lymphocytic leukemia of B-cell type (B-CLL) is a malignant disease characterized by monoclonal proliferation of small lymphocytes of B-cell origin, usually associated with suppression of polyclonal B-cell activation (i.e., proliferation and differentiation). Normal human B-cell proliferation is controlled by different T-cell-derived lymphokines, including interleukin 2 (IL2) and gamma interferon (gamma-IFN), that account for the majority of the B-cell growth factor (BCGF) activity produced by mitogen-activated peripheral blood mononuclear cells (PBMCs). We have previously shown an increased and dysregulated secretion of IL2 in peripheral blood from patients with B-CLL. BCGF, IL2, and gamma-IFN productions by phytohemagglutinin (PHA)-stimulated PBMCs were investigated in 13 patients with active untreated B-CLL and 11 healthy donors. B-CLL PBMCs produced a significant amount of BCGF (6 U/ml) despite the low percentage of T cells (10%) associated with this disease as compared with that found in healthy donors (61%). BCGF production in normal controls and B-CLL patients was tripled after irradiation of PBMCs or addition of indomethacin. gamma-IFN secretion in B-CLL patients was decreased when compared with normal controls. Therefore, when gamma-IFN was calculated per fixed number of T cells, production was significantly higher in B-CLL patients than in normal controls, showing a dilution of the productive cells. This study suggests that T cells from B-CLL patients are functional in terms of BCGF production despite their decreased percentage and abnormalities in surface markers.

Expression of receptors for IgA on mitogen-stimulated human T lymphocytes.

This study demonstrates that activation of human peripheral blood mononuclear cells (PBMC) by the T cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) induces the expression of receptors for IgA without addition of IgA to the culture medium. Cells bearing receptors for IgA were determined by indirect immunofluorescence using human secretory IgA and fluoresceinated goat anti-human IgA or goat anti-secretory component antibodies. Among freshly isolated PBMC, 4.7 +/- 1.7% of T cells, 12.7 +/- 12.5% of B cells and 14.4 +/- 7.6% of monocytes were found to be IgA receptor positive. In unstimulated PBMC cultures the percentage of IgA receptor-positive cells slightly increased at 48 h and was more elevated after 7 days. In Con A-stimulated cultures 24.3 +/- 18.5% of the cells expressed receptors for IgA after 48 h. Then, the number decreased and rose again thereafter. PHA stimulation induced an increase of smaller magnitude with similar kinetics. Induction of receptor for IgA on activated T cells was demonstrated by double-labelling experiments showing more CD8+ than CD4+ cells with receptors for IgA among Con A-activated PBMC. Furthermore, PHA or Con A stimulation of B cell-depleted PBMC suspensions resulted in a marked increase of cells bearing receptors for IgA. Expression of these receptors was down-regulated by recombinant interferon-gamma (250 units/ml) and by prostaglandin PGE2 (100 nM) both on unstimulated and mitogen-activated PBMC. The receptor for IgA was distinct from the asialoglycoprotein receptor and did not cross-react with the poly-Ig receptor of epithelial cells. It was concluded that, in the absence of inducing exogenous IgA, T cell mitogens trigger the expression of receptors for IgA. Therefore, T cell activation is associated with the down-regulation of receptors for IgM and the increased expression of receptors for IgG, IgA and IgE.

Fibroblast prostaglandin E2 synthesis. Persistence of an abnormal phenotype after short-term exposure to mononuclear cell products.

Acquired abnormalities of connective tissue metabolism in inflammatory diseases often persist when lesional tissue is maintained in in vitro culture. Although connective tissue cells are exposed to inflammatory cell-derived mediators in vivo and such mediators have been shown to alter connective tissue cell behavior, it is unclear whether the persistence of metabolic defects in vitro could result from remote in vivo exposure to these mediators. An in vitro model was used to test whether transient exposure of normal fibroblasts to inflammatory mediators could lead to metabolic alterations that persist during in vitro culture. Short-term exposure of human foreskin fibroblasts in vitro to supernates of mitogen-activated peripheral blood mononuclear cells led to persistent abnormalities of prostaglandin E2 (PGE2) metabolism. Fibroblasts previously exposed to mononuclear cell products synthesized more than twice as much PGE2 when stimulated compared with similarly stimulated but previously unexposed control fibroblasts of the same strain. The enhanced PGE2 synthesis persisted for as long as 20 wk and 19 cell generations after the original exposure to mononuclear cell products. Exposure of fibroblast populations to mononuclear cell products may, thus, lead to metabolite alterations that are still evident after multiple cell generations.

Effects of Sulindac on in vitro immunologic function and on prostaglandin production by human peripheral blood mononuclear leukocytes

Prostaglandins (PG) appear to regulate immune-mediated inflammation, but the mechanisms involved remain unclear. Several families of nonsteroidal antiinflammatory drugs (NSAID) have been observed to inhibit PG synthesis. Among these drugs, sulindac sulfide is a potent inhibitor of PG production while its parent pro-drug, sulindac sulfoxide (sulindac), lacks PG synthesis inhibitory activity in cell-free systems. We have studied the effects of sulindac sulfoxide on the blastogenic response of human peripheral blood mononuclear cells (PBMC) stimulated by exposure to alloantigens and mitogens in vitro. Sulindac inhibited proliferation of activated PBMC in a dose-dependent manner but had little effect on the proliferation of unstimulated cells. The inhibition of mitogen-induced blastogenesis correlated with both the uptake of radiolabeled drug and the inhibition of in vitro production of PG (PGE and PGF) by mitogen-activated PBMC. These data indicate a functional relationship between PG synthesis and immune cell activation which may also apply to PBMC activated in vivo by autoimmune disease. Metabolism of sulindac sulfoxide by PBMC in vitro produced too little sulindac sulfide to adequately explain the inhibition of PG production. These data suggest that immunomodulation by sulindac may be due to a direct inhibition of cellular activation. Thus, it is proposed that decreased PG production may be a result rather than the cause of the hypoproliferative response.