Human soluble IL-6 receptor: its detection and enhanced release by HIV infection.

By using a fluorescence sandwich ELISA for the quantification of soluble human IL-6R, normal human PBMC were found to be induced to release IL-6R into culture supernatant by stimulation with PHA. Furthermore, certain promonocyte cell lines and human T-cell leukemia virus I (HTLV-I)-positive cell lines produced sIL-6R into culture supernatants constitutively. However, this was not found with HTLV-I negative T cell lines and Burkitt's B cell line. In addition, generation of supernatant IL-6R of the promonocyte cell line was significantly increased 27-fold after PMA treatment and sevenfold after infection with HIV. The released IL-6R molecules were characterized as an apparent m.w. of 50 to 55 kDa by both size-exclusion HPLC and immunoprecipitation of the soluble protein with IL-6R-specific mAb followed by SDS-PAGE analysis. Furthermore, increased levels of serum IL-6R were detected in blood donors seropositive for HIV. Moreover, the released IL-6R could bind efficiently to purified rIL-6 on solid phase and suppressed the proliferative responses of PBMC. These results suggest that the release of soluble IL-6R might be linked to regulatory functions of immune responses induced by IL-6 stimulation during normal and human retrovirus-infected cell growth and differentiation.     

Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.     

Polysaccharides isolated from Açaí fruit induce innate immune responses

The Açaí (Acai) fruit is a popular nutritional supplement that purportedly enhances immune system function. These anecdotal claims are supported by limited studies describing immune responses to the Acai polyphenol fraction. Previously, we characterized γδ T cell responses to both polyphenol and polysaccharide fractions from several plant-derived nutritional supplements. Similar polyphenol and polysaccharide fractions are found in Acai fruit. Thus, we hypothesized that one or both of these fractions could activate γδ T cells. Contrary to previous reports, we did not identify agonist activity in the polyphenol fraction; however, the Acai polysaccharide fraction induced robust γδ T cell stimulatory activity in human, mouse, and bovine PBMC cultures. To characterize the immune response to Acai polysaccharides, we fractionated the crude polysaccharide preparation and tested these fractions for activity in human PBMC cultures. The largest Acai polysaccharides were the most active in vitro as indicated by activation of myeloid and γδ T cells. When delivered in vivo, Acai polysaccharide induced myeloid cell recruitment and IL-12 production. These results define innate immune responses induced by the polysaccharide component of Acai and have implications for the treatment of asthma and infectious disease.     

Inhibition of human B cell responsiveness by prostaglandin E2

The capacity of prostaglandin E2 (PCE2) to modulate human peripheral blood B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) stimulated by Cowan 1 strain Staphylococcus aureus and mitogen-stimulated T cell supernatant was examined. PGE2 significantly inhibited both responses, whereas PGF2 alpha had no inhibitory effect. Responses of highly purified B cells obtained from spleen, lymph node, and tonsil were also inhibited. In addition PGE2 suppressed B cell responses supported by recombinant interleukin 2 rather than T cell supernatant. PGE2-mediated inhibition was mimicked by forskolin, a direct activator of adenylate cyclase. Kinetic studies indicated that PGE2 inhibited B cell responses by a progressively greater increment as cultures were prolonged. Evaluation by flow cytometry after staining with acridine orange or mithramycin indicated that PGE2 had no effect on initial B cell entry into the G1 phase of the cell cycle, passage through G1, and entry into S, G2, and M. Rather, PGE2 inhibited responses of postdivisional daughter cells. PGE2 inhibited responses in cultures stimulated by the calcium ionophore ionomycin and T cell supernatant but had minimal effects in cultures stimulated by the combination of ionomycin and phorbol myristate acetate. Moreover, phorbol myristate acetate reversed PGE2-mediated inhibition of proliferation stimulated by S. aureus or S. aureus + T cell supernatant. These results indicate that PGE2 suppresses the continued growth and differentiation of human B cells, although it has no effect on initial B cell activation and suggest that PGE2 may play a role in regulating human B cell responses in vivo.     

Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.     

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

T cell activation by mycobacterial antigens in inflammatory synovitis

To define which mycobacterial antigens were responsible for the activation of synovial fluid T lymphocytes, acetone-precipitated Mycobacterium tuberculosis (AP-MT) antigens were separated into five fractions following polyacrylamide gel electrophoresis and added to the mononuclear cell cultures of patients with inflammatory synovitis. Fractions 2 (50 to 70 kDa) and 5 (less than 28 kDa) resulted in significantly more proliferation than that of fractions 1, 3, and 4. The response to a purified mycobacterial 65-kDa heat shock protein (hsp), which migrated in fraction 2, was highly correlated (r = 0.89, P less than 0.001) with the response to the crude AP-MT. The proliferative response to a different hsp. the Escherichia coli DnaK, by synovial fluid lymphocytes was marginal. Analysis of the synovial fluid T cell response to mycobacterial culture filtrates by T cell Western blotting revealed dominant responses to antigen(s) in the range of 31 to 21 kDa in each responding patient, although no other consistent pattern of T cell activation was noted. Three lines of evidence suggested that the response to the low molecular weight fractions was directed against degradation fragments of the 65-kDa protein. These observations suggest that the activation of T lymphocytes obtained from inflammatory synovial fluids by crude mycobacterial antigens was due in large part to recognition of the 65-kDa mycobacterial hsp.     

Effects of paclitaxel-producing fungal endophytes on growth and paclitaxel formation of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

Effects of a fungal endophyte, Fusarium mairei, on growth and paclitaxel formation of Taxus cuspidata cells were investigated by adding fungal endophyte culture supernatant (FECS) to suspension cultures of T. cuspidata cells. The main effective chemical responsible for paclitaxel formation in FECS was an exopolysaccharide (EPS) of molecular weight ~2 kDa. FECS fractions except EPS stimulated growth of Taxus cells but had no effects on paclitaxel accumulation. Additionally, elicitation efficiency of FECS based on different culture conditions was studied. EPS content in FECS was related to FECS culture conditions. FECS with long cultivation and high-aeration cultivation contained higher EPS content and resulted in higher paclitaxel yield than that with short cultivation and low-aeration cultivation. The maximum yield of paclitaxel from Taxus cultures, elicited by FECS with 9-day cultivation, was 4.7-fold that of the control cultures.     

Augmentation of phorbol ester-induced T cell proliferation by agents which raise intracellular cyclic adenosine monophosphate.

Although raising intracellular cyclic adenosine monophosphate (cAMP) levels is generally considered to be inhibitory on the mitogen-induced T cell proliferation, in this study we have shown that the addition of either dbcAMP (50 microM) or cholera toxin (1 ng/ml) resulted in an increase in [3H]thymidine uptake in PBMC cultures stimulated with phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA), or with a combination of TPA plus anti-CD3 mAb (mAb 235). In contrast, under similar culture conditions, the phytohemagglutinin-P (PHA-P) response was inhibited by these agents as has been reported. The augmentative effect of dbcAMP in PBMC cultures was due to an increase in IL-2 production and not to increased in IL-2R-alpha chain expression. The enhancing effect of dbcAMP and CT observed with PBMC was monocyte dependent and not seen with purified T cell preparations. The addition of monocytes reconstituted the ability of intracellular cAMP elevating agents to augment the T cell response to TPA with and without mAb to CD3. The monocytes mediate their action via soluble factor(s) with molecular weight (m.w.) of more than 10 kDa. Neither rIL-1, rIL-6, nor rTNF-alpha have any augmentative effect as contrast with the supernatant from treated monocytes. Taken together, our results indicate that cAMP can play a positive regulatory role in T cell proliferation due to factor(s) secreted by dbcAMP-treated monocytes resulting in increased IL-2 synthesis in T cells.     

Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis.

Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.     

Suppressor T cell activation by human leukocyte interferon

Murine fibroblast interferon (IFN beta) activates murine suppressor T lymphocytes in vitro, which suppress plaque-forming cell responses by spleen cells. Suppression of human in vitro immune responses by IFN was investigated to determine whether human IFN also activates suppressor T cells. Human leukocyte IFN (IFN alpha) suppressed pokeweed mitogen-induced polyclonal immunoglobulin production by human peripheral blood mononuclear cells (PBMC) by 80 to 90% at doses of 200 to 350 U/ml. Responses by IFN alpha-treated PBMC were suppressed in a dose-dependent manner; control cultures had maximal responses on day 7. PBMC incubated with 10,000 U/ml of IFN alpha contained activated suppressor cells that decreased pokeweed mitogen-stimulated, polyclonal immunoglobulin production by autologous cells by 70 to 80%. Suppression mediated by these cells was prevented by catalase, ascorbic acid, and 2-mercaptoethanol (2-ME). In murine systems, these reagents interfere with expression of suppressor T cell activity by preventing activation of soluble immune response suppressor. Selection procedures with monoclonal antibodies identified the suppressor cell as an OKT8+ (suppressor/cytotoxic) T lymphocyte. Selected OKT8+ cells required less IFN alpha (1000 U/ml) for activation and were effective in smaller numbers than unfractionated activated PBMC. IFN alpha-activated suppressor cells also inhibited proliferation in mixed lymphocyte and mitogen-stimulated PBMC cultures; again, catalase and 2-ME blocked suppression. These results indicate that IFN alpha activates suppressor T cells in human PBMC cultures; the ability of catalase, 2-ME, and ascorbic acid to block suppression suggests that these suppressor T cells have certain similarities to IFN beta or to concanavalin A-activated murine suppressor T cells.     

Comparison of the cytotoxic response against ovarian cancer by immune effector cells isolated and expanded from normal donors and ovarian cancer patients

Background/AimsThe aim of this study was to compare the cytotoxic response against ovarian cancer (OC) cells elicited by different immune effector cells in combination with the cytokines interleukin (IL)-2 and interferon (IFN) α-2b.MethodsOC cells were co-cultured with peripheral blood mononuclear cells (PBMC) from normal donors or OC patients and IL-2 or IFN α-2b alone or in combination, in order to determine the cytotoxicity. T cells were isolated from healthy donors to determine T cell cytotoxic activity. PBMC from healthy donors and OC patients were expanded in an IL-2/IL-7/IL-12 cocktail with and without anti-CD3 antibody, and the cytotoxic activity measured. Flow cytometry was performed on primary, selected and expanded cells to determine T, B, and natural killer- (NK) cell percentages.ResultsHealthy donor PBMC elicited a significant cytotoxic response (59%) compared with OC patient PBMC (7%). T cells enriched from normal donors elicited a significant cytotoxic response (18%) compared with controls lacking effector cells (1.4%); however, the cytotoxicity observed was significantly less compared with unselected PBMC. Expanded effector cells consisted primarily of T cells (98%) and the fold-expansion was significantly higher in the presence of anti-CD3 (19- versus 132-fold). No significant difference in the expansion (either fold-expansion or cell type) was observed between OC patients and healthy donors. Expanded cells from both healthy donors and OC patients elicited a significant cytotoxic response in the presence of IL-2 (19% and 22%) compared with controls.ConclusionsPBMC from OC patients do not elicit a significant cytotoxic response; however, ex vivo-expanded cells from OC patients are capable of cytotoxic killing similar to unexpanded T cells isolated from normal donors. These data provide the groundwork for further development of cellular therapy against OC.     

Gel-filtrated fractions of alveolar bone extract contain factors promoting cell attachment and a mitogenic effect on periodontal ligament fibroblasts

The purpose of this study was to investigate the effect of acetic acid-extracted bone proteins on human periodontal ligament fibroblasts (hPF) with respect to mitogenic and cell attachment promoting activity. Alveolar bone was harvested from healthy donors and subjected to 0.5 M acetic acid extraction, dialysis and lyophilization, and gel filtration. Promotion of cell attachment and stimulation of DNA synthesis by the crude extract and gel-filtrated fractions were studied in cultured hPE Many protein components, varying in molecular weight from 10-14 to 120 kDa, were detectable in 10% SDS-PAGE of the extract. Gel filtration of bone extract disclosed four fractions with molecular weights of 55, 34, 29 and 19-20 kDa. Both the 34 and 55 kDa fractions at a concentration of 5 microg/ml, but not the 29- or 19-20 kDa fractions, were found to promote cell attachment while only the 55 kDa fraction (5 microg/ml) stimulated DNA synthesis of hPF, Both mitogenic activity and the promotion of the cell attachment by gel-filtrated active fractions were resistant to thermal treatment (70 degrees C) and pH (4 to approximately 8) changes. These findings suggest that acetic acid extract of alveolar bone may contain components which are capable of modulating cell attachment and mitogenesis of hPF.     

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.     

A novel microbial interaction: obligate commensalism between a new gram-negative thermophile and a thermophilic Bacillus strain

Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated. From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60°C. The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1. In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase. Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium. For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium. The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1. During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase. Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60°C. Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp. strain SK-1. Received: November 18, 1999 / Accepted: December 2, 1999     

Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies.

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.     

Identification of T epitopes within a potential Plasmodium falciparum vaccine antigen. A study of human lymphocyte responses to repeat and nonrepeat regions of Pf155/RESA

PBMC from Melanesians who had high antibody reactivities to fusion proteins encompassing the 3' and the 5' repeat regions of the ring infected E surface antigen (Pf155/RESA), were tested for their ability to respond to synthetic and recombinant peptides representing regions of Pf155/RESA. The aim was to identify T cell epitopes within the Ag. Most of the synthetic peptides from the nonrepeat regions of Pf155/RESA were selected for study on the basis of their tendency to form amphipathic alpha-helices. Peptides representing immunodominant B cell epitopes were also tested. Three-quarters of the Melanesian donors responded to the recombinant peptides (Ag 1505 and Ag 632-100) and to the 8 x 4 mer, a synthetic peptide representative of the 3' repeat region. Whereas all the remaining eight peptides tested elicited a response in at least one donor, three peptides (M40, M42, and BTA3) representing sequences in the nonrepeat regions showed greatest promise as potentially useful T epitopes. Responses in control donors were also observed to most of the peptides but the percentage of responders was lower. T cell bulk lines specific to Ag 1505 and Ag 632-100 were established. All donors were HLA tissue typed, but no obvious correlations between responsiveness and HLA type were observed. Our results suggest that there are T cell epitopes within and outside the repeat regions of Pf155/RESA.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.     

Role of adherent cells in dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation in the rat and have found that the addition of the compound Dextran sulfate (DxS), which itself is not mitogenic, to LPS stimulated cultures results in significant enhancement of cell division. A "DxS-free" supernatant from DxS stimulated spleen cell cultures is able to substitute for DxS in stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity, however, the addition of purified recombinant IL-1 to LPS-stimulated cultures does not result in augmentation of proliferation. A DxS-free supernatant from DxS stimulated adherent cells is also able to substitute for DxS in stimulatory activity. The active molecule(s) present in the adherent cell-derived DxS-free culture supernatant appears to be distinct from classical IL-1.     

The purification and biological activity of a macrophage-derived factor with interleukin 1-like activities from guinea pigs

A macrophage-derived factor with interleukin 1-like activity was purified from culture supernatant of muramyl dipeptide-stimulated peritoneal exudate macrophages of guinea pigs. Starting with serum-free culture supernatant, the purification was carried out by gel permeation chromatography, affinity chromatography on procion red agarose, removal of carry-over serum proteins by Sepharose-coupled antibodies against bovine serum proteins, anion exchange chromatography and hydrophobic chromatography. The purified sample potentiated the phytohemagglutinin-induced thymidine uptake of thymocytes with a 50% effective concentration of 9.6 X 10(-11) M. The sample showed a single band in polyacrylamide gel electrophoresis, and a 65 kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis by silver staining. A single peak of activity was detected by thymocyte assay at the position corresponding to the stained band in both of the electrophoretic analyses. The purified factor had activities to potentiate the antigenic activation of sensitized T cells for the production of a lymphokine, macrophage migration inhibitory factor, and also the proliferative response of sensitized T cells to antigen. Thus, the 65 kDa factor has activities to modulate various T cell responses in guinea pigs such as interleukin 1 does in other species. The molecular relationship of the 65 kDa macrophage factor to interleukin 1 remains to be determined.