Unstimulated Control Cultures Research Articles

Capsaicin-induced activation of ERK1/2 and its involvement in GAP-43 expression and CGRP depletion in organotypically cultured DRG neurons.

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.

Dose Dependent Effects of STAT5 on Proliferation, Differentiation and Self Renewal of Hematopoietic Stem/Progenitor Cells.

To study the dose dependent effects of ectopically expressed STAT5A in cordblood (CB) CD34+ cells, we developed a tamoxifen-inducible STAT5A expression system. Constitutively active STAT5A (STAT5A1*6) was fused to the ER domain to facilitate its activation upon stimulation of the transduced cells by the addition of 4OHT. Transactivation and DNA binding studies revealed that STAT5A could be activated to different levels in a controlled way by addition of different concentrations of 4OHT. CB CD34+ cells were retrovirally transduced with this construct and the effects on proliferation, differentiation and self renewal was studied.In an MS5 coculture system, we demonstrated that a high dose of 4OHT (500nM) resulted in a rapid burst of erythropoiesis and a block in myeloid differentiation (as previously demonstrated using STAT5A1*6 without an ER domain). Moderate levels of 4OHT (20nM) however, resulted in a much higher proliferation of not yet differentiated cells, ultimately resulting in a higher expansion. This higher expansion coincided with a higher frequency of early (day10) cobblestones (without 4OHT: 1,5 cobblestones per 104 plated cells, 20nM 4OHT: 1750/104, 500nM 4OHT:200/104). RT-PCR of STAT5A1*6-ER transduced cells revealed a rapid downregulation of C/EBPalpha mRNA upon stimulation with 4OHT, with a dose response curve inversely correlated to the number of early cobblestones, suggesting that these two processes are linked.When STAT5A1*6-ER transduced cells were cultured in methylcellulose, stimulation with increasing concentrations of 4OHT resulted in decreasing numbers of colonies (myeloid as well as erythroid). Interestingly, whereas colonies from unstimulated (control) cultures and 500nM 4OHT stimulated cultures could not be secondary plated, cultures stimulated with only very low concentrations of 4OHT (1 nM) could be secondary plated, giving rise to only myeloid colony formation. These findings demonstrate that the level of activation of STAT5 affects the proliferation and differentiation in a separate manner; low levels of STAT5A activation results in self renewal of the progenitor/stem cell pool cells, whereas high levels of STAT5A induces a rapid differentiation at the expense of proliferation and self renewal.These data correspond well to our findings in AML cells. Downregulation of the moderate constitutive STAT5 activation in CD34+ AML cells by STAT5 RNAi affects especially the proliferative capacity of the AML cells. In conclusion, we hypothesise that low constitutive levels of active STAT5 might contribute to leukemic transformation.

Decreased CD90 expression in human mesenchymal stem cells by applying mechanical stimulation

BackgroundMesenchymal stem cells (MSC) are multipotent cells which can differentiate along osteogenic, chondrogenic, and adipogenic lineages. The present study was designed to investigate the influence of mechanical force as a specific physiological stress on the differentiation of (MSC) to osteoblast-like cells.MethodsHuman MSC were cultured in osteoinductive medium with or without cyclic uniaxial mechanical stimulation (2000 μstrain, 200 cycles per day, 1 Hz). Cultured cells were analysed for expression of collagen type I, osteocalcin, osteonectin, and CD90. To evaluate the biomineral formation the content of bound calcium in the cultures was determined.ResultsAfter 14 days in culture immunfluorescence staining revealed enhancement of collagen type I and osteonectin expression in response to mechanical stimulation. In contrast, mechanically stimulated cultures stained negative for CD90. In stimulated and unstimulated cultures an increase in the calcium content over time was observed. After 21 days in culture the calcium content in mechanical stimulated cultures was significantly higher compared to unstimulated control cultures.ConclusionThese results demonstrate the influence of mechanical force on the differentiation of human MSC into osteoblast-like cells in vitro. While significant enhancement of the biomineral formation by mechanical stimulation is not detected before 21 days, effects on the extracellular matrix became already obvious after 14 days. The decrease of CD90 expression in mechanically stimulated cultures compared to unstimulated control cultures suggests that CD90 is only transiently expressed expression during the differentiation of MSC to osteoblast-like cells in culture.

T-Cell-Stimulating Protein A Elicits Immune Responses during Meningococcal Carriage and Human Disease

In recognition of the need for immunological memory-inducing components for future Neisseria meningitidis group B vaccines, we previously searched the proteome of N. meningitidis and identified T-cell-stimulating protein A (TspA). This study was designed to confirm the immunogencity of TspA and to examine the subset of T-helper cell responses to the protein in patients and nasopharyngeal carriers. The tspA gene was reconstructed, cloned, and expressed in Escherichia coli, and the recombinant TspA (rTspA) protein was affinity purified. T-cell proliferative responses to rTspA were detected in the peripheral blood mononuclear cells (PBMCs) of convalescent patients and carriers, confirming that TspA-specific T-cell responses were stimulated by invasive disease and nasopharyngeal colonization. Following stimulation of PBMCs with meningococcal lysate, increased frequencies of both Th1 and Th2 cells were observed, indicating that, as during carriage, invasive meningococcal disease induced an unbiased T-helper subset response. A similar unbiased T-helper response was also detected against rTspA in the PBMCs of convalescent patients. The response of PBMCs from the carriers to TspA stimulation, however, was very weak, and the frequencies of cytokine-positive CD4 cells were not significantly greater than the frequencies in unstimulated control cultures. All of the patients and carriers responded with serum antimeningococcal immunoglobulin G (IgG) antibodies, while four of six samples from patients and 5 of 14 samples from carriers contained detectable anti-rTspA IgG antibodies. Taken together, the results of this study confirmed the immunogenicity of TspA in humans during natural meningococcal infection, and therefore, TspA is worthy of further investigation as a possible T-cell stimulating component of future vaccines.

Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro

This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24 h of induction and cytokine mRNA expression was detected using RT-PCR at 6 h post induction. For IFN-α/β it was found that both plasmid DNA and phosphodiester ODN, containing an unmethylated CpG-motif, were able to induce IFN production in the presence of lipofectin but not without. The levels of IFN varied with individuals and were often quite low. Moreover, methylation or removal of the CpG sequence completely abolished IFN induction. CpG-containing ODN with poly-guanine (G) sequences in the 5′ and 3′ ends induced considerably higher levels of IFN, especially when the poly-G sequences had a phosphorothioate backbone. ODN with poly-G sequences also had the ability to induce IFN in the absence of lipofectin but the levels of IFN induced were radically reduced compared to those induced with lipofectin. In contrast to IFN, which was only detected upon induction, low spontaneous IL-6 production was observed in unstimulated control cultures. Nevertheless, plasmid DNA and CpG-containing ODN were able to increase the IL-6 production threefold. All the IFN inducing ODN also induced IL-6 production and the levels of IL-6 induced seemed influenced by addition of lipofectin and presence of poly-G sequences in the same way as was observed for the IFN-production. However, a complete phosphorothioate ODN with a central CpG-motif and poly-C sequences, that did not induce IFN, readily induced IL-6 both in the presence and absence of lipofectin. In addition, there was also evidence that some ODN induced increased expression of IL-12p40 mRNA. To conclude, equine PBMC were able to recognize CpG-DNA and respond with both IFN-α/β and/or IL-6 production. The levels of cytokine induced, and sometimes which cytokine induced, varied with, e.g., CpG-motifs used, the presence of poly-G sequences, ODN backbone chemistry and presence of lipofectin.

Extracellular matrix synthesis, proliferation and death in mechanically stimulated human gingival fibroblasts in vitro

Gingival tissues are constantly exposed to the effect of physical forces. Mechanical stimuli are regulators of connective tissue homeostasis and sustained mechanical stimulation may lead to modifications in cell activity and extracellular matrix (ECM) composition. This study examined in vitro ECM synthesis, proliferation, and death in mechanically stimulated human gingival fibroblastlike cells. Four primary human cell strains were established and subjected to intermittent stretching in FX-3000 Flexercell Strain Unit for 10 days, 45 min/day, at 1 Hz, 10% strain, and cell proliferation, cell death, and synthesis of collagens types I, III, and V, matrix metalloproteinase 1, elastin, and tenascin were assessed. In some of the cell strains mechanical stimulation led to changes in synthesis of the ECM molecules, proliferative activity, and death of stimulated cells as shown by statistically significant differences between the experimental and unstimulated control cultures. Although not seen in every culture investigated, these findings suggest that prolonged mechanical stimulation might lead to conspicuous modifications in the metabolic activity of gingival fibroblasts and cause changes in the ECM composition of the gingival connective tissue. The results indicate a pronounced interindividual variation in reactions of gingival fibroblasts to mechanical stimulation.

Immunoregulation in onchocerciasis: predominance of Th1-type responsiveness to low molecular weight antigens of Onchocerca volvulus in exposed individuals without microfilaridermia and clinical disease.

Chronic and generalized onchocerciasis is associated with suppression of the parasite-specific cellular responsiveness, while exposed individuals without parasitological and clinical evidence of infection (endemic normals) display prominent cellular reactivity to Onchocerca volvulus antigens (OvAg). In order to identify those parasite antigens which may account for this differential cellular responsiveness, total adult worm-derived OvAg were fractionated by means of preparative SDS-PAGE and blot-elution into 22 antigen fractions of continuously decreasing molecular weight. Peripheral blood mononuclear cells (PBMC) from microfilariae (mf)-positive onchocerciasis patients (n = 18) proliferated weakly in response to all OvAg fractions. In contrast, in vitro reactivity of PBMC from endemic normals (n = 9) was depressed in response to OvAg of mol. wt 200-30 kD only, while antigens of mol. wt < 30 kD induced vigorous proliferation in these individuals compared with the microfilaridermic patients (P < 0.05). Highest proliferative reactivity of cells from endemic normals was observed in response to OvAg of mol. wt 15-11 kD. Furthermore, these low mol. wt antigen fractions induced substantial production of IL-2 and interferon-gamma (IFN-gamma) in PBMC from endemic normals, but not in those from onchocerciasis patients. Cells from individuals of both groups secreted similar amounts of IL-5 in response to all OvAg fractions, with highest production again being induced by low mol. wt OvAg. In contrast, PBMC from onchocerciasis patients clearly produced more IL-10 than did cells from endemic normals. This augmented IL-10 production by PBMC from mf-positive individuals was not only observed after stimulation with OvAg fractions, but was measured in unstimulated control cultures as well. IFN-gamma-specific mRNA in antigen-stimulated PBMC from endemic normals appeared to be more prominent than in cells from onchocerciasis patients. However, mRNA transcripts of IL-10 and IL-13 were clearly present in patients, but were absent or inconsistently observed in endemic normals. Our results suggest that vigorous Th1-type cellular responsiveness encountered in endemic normals is restricted to low mol. wt antigens of O. volvulus, while such reactivity will not be present in mf-positive individuals. Furthermore, spontaneous production of high levels of IL-10 in onchocerciasis patients is likely to suppress Th1-type immunity, and thus may favour manifestation of chronic onchocerciasis. These traits of cellular immunity may contribute to the differential outcome of O. volvulus infection, the manifestation of clinical disease, and may also regulate the build up of acquired immunity in humans.

Role of cyclical pressure and particles in the release of M-CSF, chemokines, and PGE2 and their role in loosening of implants.

A septic loosening of orthopaedic implants is usually attributed to the action of wear debris from the prosthesis. Recent studies, however, have also implicated physical pressures in the joint as a further cause of loosening. We have examined the role of both wear debris and pressure on the secretion of two chemokines, MIP-1alpha and MCP-1, together with M-CSF and PGE2, by human macrophages in vitro. The results show that pressure alone stimulated the secretion of more M-CSF and PGE2 when compared with control cultures. Particles alone stimulated the secretion of M-CSF and PGE2, when compared with unstimulated control cultures, but did not stimulate the secretion of the two chemokines. Exposure of macrophages to both stimuli simultaneously had no synergistic effect on the secretion of the chemokines, but both M-CSF and PGE2 were increased in a synergistic manner. Our findings suggest that pressure may be an initiating factor for the recruitment of cells into the periprosthetic tissue.

Proliferation responses in preimmunized mice lymphocytes by Bordetella pertussis cell wall components.

Bordetella pertussis infects the respiratory tract of the human host and causes whooping cough in children. The nature of immunity against Bordetella pertussis infection and disease is poorly understood. The aim of this study was to investigate cell mediated immunity in mice immunized with outer membrane component of cell wall, of B. Pertussis. A group of mice were immunized with outer membrane complex (OMC) and killed whole cell (WCV) of B. pertussis, with an interval of 2 weeks. During a period of 7 weeks following the immunization, lymphocytes were isolated from lymph nodes of immunized mice. The in vitro proliferative response of isolated lymphocyte to stimulation with 20 ig of 30 and 69 kDa outer membrane protein (OMP) were measured as parameters for cell mediated immunity (CMI). The data were expressed as mean count per minute (CPM)x103 after subtraction of the CPM of unstimulated control cultures. Lymphoblastogenic response was observed in immunized mice with WCV and OMC. At 30 days of post immunization a significant increase in response to 30 and 69 kDa OMP was observed, a small decrease in the response was evident against P30 and P69 at 60 and 120 days of post immunization, but the response was still higher than what was observed in control mice. Current findings indicate strongly the potential of outer membrane protein component of B. perlussis in proliferating lymphocytes in the mice.

Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells.

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.

Activation of phospholipase D signaling pathway by epidermal growth factor in osteoblastic cells.

The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 microCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10-60 s) and long (up to 30 minutes) incubation periods (p < 0.05). Phosphatidic acid levels were also significantly increased (p < 0.05) compared with unstimulated controls, but the levels were approximately 60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.

Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-γ is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.

Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-gamma is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.

Regulation of NK cell functions by TGF-beta 1.

The contribution of exogenous and endogenous TGF-beta 1 to human peripheral blood NK cell proliferation and activity in vitro was investigated. Exogenous bioactive TGF-beta 1 inhibited NK cell DNA synthesis and production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) by NK cultures consisting of > 98% CD56+ cells. The cytotoxic activity of NK cells was also weakly inhibited by exogenous TGF-beta 1. All TGF-beta 1-induced inhibitory effects occurred in the absence and presence of the NK cell-activating cytokines IFN-alpha, IL-2, and IL-12. Unstimulated NK cell cultures expressed steady state TGF-beta 1 mRNA detected by Northern blot analysis and produced TGF-beta protein (1.6 ng/ml), as determined by ELISA. When NK cell proliferation was induced by IL-2, IL-12, IFN-alpha, or a combination of IL-2 and IL-12, expression of TGF-beta 1 mRNA and protein was moderately and consistently reduced by approximately 20%, as compared with unstimulated control cultures. Unstimulated and rapidly proliferating NK cell cultures secreted primarily latent TGF-beta into their culture medium, as determined by the Mv1Lu bioassay. These results indicate that, in vitro, endogenous NK cell-derived TGF-beta 1 has no negative autocrine effect upon activation of NK cells by various cytokines.

Effects of Pertussis Toxin on Resorption of 19-Day-Old Fetal Rat Long Bones*

We tested the effects that pertussis toxin had on bone resorption mediated by cAMP-dependent and cAMP-independent stimuli in 19-day-old fetal rat long bones. Agents that stimulate cAMP were PTH, prostaglandin E2, and calcitonin. Agents that act independent of cAMP were: phorbol 13-myristate 12-acetate (PMA), 1,25-dihydroxyvitamin D3, murine interleukin-1 alpha, osteoclast-activating factor, and human tumor necrosis factor-alpha. Pertussis (1-10 ng/ml) produced a dose-related inhibition of resorption in unstimulated control cultures. The inhibitory effect was not associated with changes in either [3H]thymidine or [3H]proline incorporation into bones. beta-Glucuronidase activity in the medium was decreased. PMA was the only agonist whose resorptive effect was completely blocked by pertussis. The resorptive response to other stimulators was reduced, but treated/control ratios usually remained the same or increased because of the greater effect of pertussis on control resorption. There was a partial inhibition of the resorptive effect of low doses of prostaglandin E2 (10 nM), but increasing the concentration of agonist overcame the inhibition. Pertussis did not enhance the sensitivity of bones to calcitonin. Pertussis enhanced the cAMP response to PTH, but had no effect on basal cAMP production. Since PMA was inhibited by pertussis while agents that may act through cAMP-mediated or phosphatidylinositol pathways were not affected, we hypothesize that a protein kinase-C dependent pathway can modulate bone resorption.

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1-stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.

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.

Retinal S antigen reactivity in patients with retinitis pigmentosa and Usher's syndrome.

Cell-mediated immunity to retinal antigens as determined by in vitro assays has been detected in a variety of retinal degenerative and retinal destructive conditions. The authors studied the in vitro reactivity to the soluble retinal S antigen of peripheral blood mononuclear cells from patients with simplex and multiplex retinitis pigmentosa (RP) and Usher's syndrome along with normal controls. The cellular stimulation index (SI) was calculated by comparing 3H-thymidine incorporation in cells exposed to either purified S antigen, crude whole human retinal antigen, or human choroidal antigen with that in unstimulated control cultures. Eight of 15 patients with RP and 5 of 6 with Usher's had a SI greater than the tolerance limits of normal values. These results indicate that the immune system may be involved either primarily or secondarily in RP.

In vitro viability of lymphoid cells from lines of mice genetically selected for high or low responsiveness to phytohemagglutinin

The kinetics of viability of lymph node and spleen cells of mice genetically selected for “high” or “low” in vitro lymphocyte responsiveness to PHA were studied in PHA or PPD-stimulated short-term cultures. Lo/PHA cells were found to be less viable than Hi/PHA cells in unstimulated control cultures. PHA improved the viability of Lo/PHA cells while inducing proliferation of Hi/PHA cells with the appearance of more and larger lymphoblasts in the latter. PPD only improved the viability of spleen cell cultures, more so for the Hi/PHA line. The interline difference in thymidine uptake was smaller after PPD than after PHA stimulation. Modifications of culture conditions designed to decrease the interline difference in cell viability lessened but did not abolish the separation between the two lines for the PHA response as measured by thymidine uptake.

Similar disturbances in B cell activity and regulatory T cell function in Henoch-Schönlein purpura and systemic lupus erythematosus.

The immunoglobulin synthesizing activities of peripheral mononuclear cells (MNC) from five patients with Henoch-Schonlein purpura (HSP) and eight patients with active systemic lupus erythematosus (SLE) were compared. Cumulative amounts of IgM, IgG, and IgA synthesized and secreted by unstimulated and PWM-stimulated patient cells over a 12-day period were determied in a solid-phase radioimmunoassay. In unstimulated control cultures mean rates of IgM, IgG, and IgA synthesis were less than 250 ng/ml. The synthetic activities of patient MNC were markedly increased. In HSP cultures IgA was the major immunoglobulin class produced (2810 x/divide 1.33 ng/ml) followed by IgG (1754 x/divide 1.32 ng/ml) and IgM (404 x/divide 1.16 ng/ml). In SLE cultures IgA and IgG syntheses were equally elevated (4427 x/divide 1.20 and 4438 x/divide 1.49 ng/ml, respectively) whereas IgM synthesis averaged 967 x/divide 1.66 ng/ml. PWM stimulation of pateient MNC caused a sharp decline in the synthesis of all three immunoglobulin classes. After T cell depletion B cell-enriched fractions from HSP and SLE patients maintained high levels of IgA and IgG synthesis that were inhibited by PWM and by normal allogeneic but not autologous T cells. In PWM-stimulted co-cultures, patient T cells nonspecifically suppressed the synthetic activities of autologous and control Bmore » cells. in contrast patient B cells achieved normal levels of immunoglobulin synthesis when cultured with control T cells plus PWM. In longitudinal studies patient B and T cell disturbances persisted despite clinical improvement.« less