Histamine regulates lymphocyte mitogenic responses through activation of specific H1 and H2 histamine receptors.

In previous studies we have reported that patients with mild atopic eczema have enhanced lymphocyte mitogenesis while those with severe disease have markedly suppressed responses. Similarly, histamine in low concentrations enhanced mitogenesis while higher levels inhibit mitogen stimulated thymidine uptake. In the present study, we investigated the kinetics of this response and the interaction of histamine with its cell-surface receptors on lymphocytes. Histamine (10(-3) M) markedly inhibited [3H]-thymidine incorporation to 27% of control levels when added at the beginning of a 72 h culture period. When added after 24 and 48 h of culture, however, the suppression was much less (62 and 88% of control). Lymphocyte cultures pulsed for 1 h with histamine, washed free of the agent and then cultured with mitogen also showed marked suppression of [3H]-thymidine uptake. The kinetics of the response suggest that histamine acts to inhibit initial processing or recruitment steps in the mitogenic assay. Cimetidine, an H2-receptor blocking agent, prevented the suppressive effect of high levels of histamine while diphenhydramine, an H1 blocker, abolished the enhancement observed with low levels. Pre-incubation of mononuclear cell suspensions, which has been shown to decrease suppressor activity, resulted in a decreased response to added histamine. This change in histamine responsiveness was associated with an alteration in H1:H2 histamine binding as determined with a radiolabelled ligand-binding assay. Histamine suppression of mitogenesis was associated with an increase in cellular cAMP levels while enhancement was accompanied by a small increase in cGMP. These data suggest that lymphocyte function may be regulated, in part, by histamine receptor bearing cells with H1 stimulation having a role in enhancement of mitogenesis and H2 stimulation resulting in normal suppressor activity.     

Tritiated-thymidine uptake in mixed leucocyte cultures: effect of specific activity and exposure time

The degree of in vitro transformation of lymphocytes to blast cells can be estimated by measuring the uptake of radioactive precursors into DNA. We have used ³H-thymidine uptake to quantitate blastogenesis in mixed leucocyte cultures. In experiments designed to standardize this procedure, the kinetics of thymidine uptake were studied by adding 4 μCi of ³H-thymidine of varying specific activities to 5-day mixed cultures. With high specific activity (0·194 μg total thymidine/culture), the rate of uptake was constant for only about 6 hr, then declined. There was no further cellular uptake between 8 and 24 hr, even though the total radioactivity of the supernatant medium did not diminish appreciably. This decreasing rate of uptake was at least partly the result of thymidine degradation to thymine and dihydrothymine by cellular enzymes. It is also possible that eventual radiation damage to blast cell nuclei may have retarded DNA synthesis when ³H-thymidine specific activity was high. With decreasing specific activity (up to 19·4 μg thymidine/culture), the rate of uptake became more nearly linear throughout 24 hr exposure to the isotope. The possible effects of thymidine degradation and radiation damage should be considered when measuring radioactive thymidine uptake in vitro, and short labelling times should be used whenever feasible.     

Conjugation of hydroxyethyl starch to desferrioxamine (DFO) modulates the dual role of DFO in Yersinia enterocolitica infection

The iron chelator desferrioxamine (DFO) B is widely used in the therapy of patients with iron overload. As a side effect, DFO may favor the occurrence of fulminant Yersinia infections. Previous work from our laboratory showed that this might be due to a dual role of DFO: growth promotion of the pathogen and immunosuppression of the host. In this study, we sought to determine whether conjugation of DFO to hydroxyethyl starch (HES-DFO) may prevent exacerbation of Yersinia infection in mice. We found HES-DFO to promote neither growth of Yersinia enterocolitica nor mitogen-induced T-cell proliferation and gamma interferon production by T cells in vitro. Nevertheless, in vivo HES-DFO promoted growth of Y. enterocolitica possibly due to cleavage of HES and release of DFO. The pretreatment of mice with DFO resulted in death of all mice 2 to 5 days after application of a normally sublethal inoculum of Y. enterocolitica, while none of the mice pretreated with HES-DFO died within the first 7 days postinfection. However, some of the HES-DFO-treated mice died 8 to 14 days postinfection. Thus, due to the delayed in vivo effect HES-DFO failed to trigger Yersinia-induced septic shock, which accounts for early mortality in DFO-associated septicemia. Moreover, our data suggest that DFO needs to be taken up by host cells in order to exert its immunosuppressive action. These results strongly suggest that HES-DFO might be a favorable drug with fewer side effects than DFO in terms of DFO-promoted fulminant infections.     

Reversion of age-related recognition memory impairment by iron chelation in rats

It is now generally accepted that iron accumulates in the brain during the ageing process. Increasing evidence demonstrate that iron accumulation in selective regions of the brain may generate free radicals, thereby possessing implications for the etiology of neurodegenerative disorders. In a previous study we have reported that aged rats present recognition memory deficits. The aim of the present study was to evaluate the effect of desferoxamine (DFO), an iron chelator agent, on age-induced memory impairment. Aged Wistar rats received intraperitoneal injections of saline or DFO (300mg/kg) for 2 weeks. The animals were submitted to a novel object recognition task 24h after the last injection. DFO-treated rats showed normal recognition memory while the saline group showed long-term recognition memory deficits. The results show that DFO is able to reverse age-induced recognition memory deficits. We also demonstrated that DFO reduced the oxidative damage to proteins in cortex and hippocampus. Thus, the present findings provide the first evidence that iron chelators might prevent age-related memory dysfunction.     

Conjugation of Hydroxyethyl Starch to Desferrioxamine (DFO) Modulates the Dual Role of DFO in Yersinia enterocolitica Infection

The iron chelator desferrioxamine (DFO) B is widely used in the therapy of patients with iron overload. As a side effect, DFO may favor the occurrence of fulminant Yersinia infections. Previous work from our laboratory showed that this might be due to a dual role of DFO: growth promotion of the pathogen and immunosuppression of the host. In this study, we sought to determine whether conjugation of DFO to hydroxyethyl starch (HES-DFO) may prevent exacerbation of Yersinia infection in mice. We found HES-DFO to promote neither growth of Yersinia enterocolitica nor mitogen-induced T-cell proliferation and gamma interferon production by T cells in vitro. Nevertheless, in vivo HES-DFO promoted growth of Y. enterocolitica possibly due to cleavage of HES and release of DFO. The pretreatment of mice with DFO resulted in death of all mice 2 to 5 days after application of a normally sublethal inoculum of Y. enterocolitica, while none of the mice pretreated with HES-DFO died within the first 7 days postinfection. However, some of the HES-DFO-treated mice died 8 to 14 days postinfection. Thus, due to the delayed in vivo effect HES-DFO failed to trigger Yersinia-induced septic shock, which accounts for early mortality in DFO-associated septicemia. Moreover, our data suggest that DFO needs to be taken up by host cells in order to exert its immunosuppressive action. These results strongly suggest that HES-DFO might be a favorable drug with fewer side effects than DFO in terms of DFO-promoted fulminant infections.     

Resistance of pokeweed mitogen-stimulated B cells to inhibition by deoxyadenosine.

Deoxyadenosine (dAdo) levels above 2 microM inhibit plasma cell (PC) differentiation by human blood lymphocytes in pokeweed mitogen (PWM) stimulated cultures containing deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase (ADA). ADA inhibition by dCF alone did not suppress PC differentiation. Thymidine uptake by T cell blasts continuously cultured in conditioned medium was inhibited by dAdo and dCF; two of five EBV-infected B cell lines were also inhibited while three were resistant. Inhibition of PWM-induced PC differentiation of B cells by dCF and dAdo was reversed when conditioned medium (a source of T cell helper factors) was added to the cultures, and dAdo and dCF added to PWM-stimulated cultures 48 hr after their initiation did not inhibit PC differentiation, though thymidine uptake and the total number of cells recovered from the cultures were reduced. Removal of T cells after 48 hr of culture slightly reduced the numbers of PC in PWM-stimulated lymphocyte cultures but no further inhibition was obtained when dCF and dAdo were added to these T-depleted cultures, nor was their thymidine uptake further reduced. These results suggest that the in vitro suppression of B cell differentiation by dAdo in PWM-stimulated cultures is not due to direct toxicity of purine nucleosides to B cells but may be due to interference with T cell help. This is consistent with the view that a relative lack of helper activity by T cells contributes to the antibody deficiency of patients with ADA deficiency.     

Cimetidine abrogates suppressor T cell function in vitro

Cimetidine increased the [3H] thymidine incorporation of normal human mononuclear cells in culture both when unstimulated or when under the stimulus of phytohemagglutinin or pokeweed mitogen (PWM). It also increased their supernatant immunoglobulin production under PWM stimulus. These effects were higher when the cells were preincubated with cimetidine than when it was added simultaneously. To determine if this effect of cimetidine reflects an abrogation of suppression we studied concanavalin-A-induced suppressor function of normal mononuclear cells using both [3H] thymidine incorporation and immunoglobulin synthesis as indicator systems and found that preincubation with cimetidine caused significant decrease in suppressor cell function in both systems.     

Cyclosporin A directly inhibits human B-cell proliferation by more than a single mechanism

Cyclosporin A (CsA) is a potent immunosuppressive agent that inhibits T-cell proliferation and lymphokine production. There is less information on the direct effect of CsA on B-cells. We investigated the proliferative responses of human tonsillar B-lymphocytes to a "T dependent" mitogen, pokeweed mitogen (PWM), and to a "T independent" mitogen, Staphylococcus aureus (SA). Both responses were strongly inhibited by CsA. Nonspecific cytotoxicity was ruled out, and the inhibition was not reversed by adding IL1, IL2, or BCGF individually or in combination. Maximal inhibition of the PWM response occurred when CsA was added early in the culture period. Cyclosporin A added 18 hours after the start of culture was less effective, and adding CsA after 36 hours resulted in only minimal inhibition. However, with SA as mitogen, addition after 36 hours still affected substantial inhibition. These results, on the time of action and resistance to reversal by exogenous growth factors, suggest that CsA can directly inhibit human B-cells by a mechanism similar to its action on T-lymphocytes, blocking an early event critical to entry into cell cycle, but an additional mechanism of inhibition later in the cell cycle may also operate when the proliferative signal is provided by the T-independent mitogen SA.     

Deoxycytidine reverses inhibition of morphogenesis by thymidine in young chick blastoderm

Exogenous thymidine affects morphogenesis of the early chick blastoderm possibly by depleting the deoxycytidine triphosphate pool. The aim of this study is to determine whether the inhibitory action of thymidine on early chick blastoderm morphogenesis is alleviated by the removal of thymidine and/or treatment with deoxycytidine. Chick blastoderms at the full hypoblast stage develop abnormally in egg albumen containing 1.23 X 10(-3) M thymidine. Development is normal when deoxycytidine is included simultaneously in the culture medium with thymidine at equimolar concentrations. Blastoderms were cultured in egg albumen containing 15 microCi/ml thymidine [methyl-3H] or 10 microCi/ml deoxycytidine [5-3H], and 1.2 X 10(-3) M 2'-deoxycytidine or 1.23 X 10(-3) M thymidine, respectively. The culture was interrupted at timed intervals, and the amount of radioactivity associated with DNA was determined. Exogenous deoxycytidine in the culture medium caused a noticeable increase in the incorporation of 3H-thymidine, while exogenous thymidine markedly inhibited the uptake and incorporation of 3H-deoxycytidine into DNA of blastoderms. Thymidine does not inhibit the expansion of blastoderm, the migration of cells for formation of the primitive streak (PS), and the induction of axial tissues, but it interferes with the organization of these tissues to form the embryonic axis. Blastoderms show slight signs of recovery when thymidine is removed. Deoxycytidine counteracts the action of thymidine and seems to be a rate-limiting factor in normal differentiation of the early chick blastoderm.     

Effects of non-steroidal anti-inflammatory drugs on lymphocyte activation

Guinea-pig lymph node lymphocytes were stimulated with mitogen (phytohaemagglutinin)in vitro and lymphocyte activation was measured by tritiated thymidine incorporation (DNA synthesis). Inclusion of non-steroidal anti-inflammatory drugs (NSAIDs) in the culture medium at therapeutic concentrations, frequently exerted an inhibitory effect. Such inhibition could be not attributed to the ability of these drugs to inhibit cyclo-oxygenase or lipoxygenase enzymes. Inhibition by salicylates was not associated with cytotoxic or cytopathic effects, since inhibition was only evident when the drugs were included in the early phase of culture. Other NSAIDs exhibited varying degrees of toxicity, which in some instances may account for observed inhibition. The effects on lymphocyte activation of selective inhibitors of pathways of arachidonic acid metabolism, do not support the proposition that the generation of prostaglandins, thromboxanes, leukotrienes or related compounds is an obligatory step during lymphocyte activation.     

Regulation of mitogen- and antigen-stimulated lymphocyte blastogenesis by prostaglandins.

Exogenously added prostaglandin E1 or E2 inhibited the blastogenic response of Mycobacterium bovis-sensitized bovine peripheral blood lymphocytes stimulated with concanavalin A, phytohemagglutinin, or M. bovis purified protein derivative as measured by [3H]thymidine uptake. The kinetics of the response showed that prostaglandins must be added to lymphocyte cultures within hours after mitogen or antigen addition to achieve maximum suppression of [3H]thymidine uptake. Addition of prostaglandins 24 h after the addition of mitogens or antigens resulted in considerably less suppression, supporting a hypothesis that prostaglandins initiate an early series of events which ultimately control lymphocyte blastogenesis rather than directly inhibit deoxyribonucleic acid synthesis.     

The production of lymphocyte mitogenic factor and migration–inhibition factor by antigen-stimulated lymphocytes of subjects with grass pollen allergy

Peripheral blood lymphocytes of twenty-three subjects with grass pollen allergy were cultured with grass pollen antigen for 3 days. After harvesting, the culture supernatants were added to fresh autologous lymphocytes which were maintained in culture for 6 days. Cellular uptake of [³H]thymidine was measured during the sixth day of culture, and revealed that the lymphocyte culture supernatants stimulated greater thymidine uptake than expected from the lymphocyte transformation response to corresponding amounts of antigen. The supernatant factor which mediated this effect was termed `lymphocyte mitogenic factor' by analogy with a similar response of lymphocytes in clinical and experimental delayed hypersensitivity. Lymphocyte culture supernatants were also tested for migration–inhibition factor by their ability to inhibit the migration of guinea-pig macrophages.

The majority of `allergic' supernatants contained a lymphocyte mitogenic factor active at 1/3 dilution (14/22) and 1/12 dilution (19/21) in contrast to supernatants derived from non-allergic subjects (2/16 and 1/17 respectively). The production of lymphocyte mitogenic factor corresponded to the occurrence of antigen-induced lymphocyte transformation (allergic: 18/22; non-allergic: 1/14); but only a minority of allergic supernatants contained a migration–inhibition factor (6/20). Clinical analysis revealed that migration–inhibition factor was particularly associated with the milder forms of allergy and with a past history of desensitization by depot injection of emulsified pollen antigen. In contrast, lymphocyte transformation and the production of mitogenic factor were uniformly distributed among the various categories of allergic subjects, all of whom had immediate (reaginic) hypersensitivity, but only three of whom had delayed hypersensitivity.

The demonstration of lymphocyte mitogenic factor in a clinical state dominated by immediate hypersensitivity supported the view that antigen-induced transformations are generally mediated by soluble `mitogenic' factors and that such mediators are not necessarily identical to those which can inhibit macrophage migration. It appeared that some features of cellular immunity were associated with grass pollen allergy, in which it is suggested that heterogeneity of lymphocyte-derived mediators may underlie an apparent dissociation of cellular immune functions. On this basis it is proposed that clinical expression of an atopic state may be partly governed by which features of cellular immunity are present.

     

The BrdU content of DNA is decreased during reversal of inhibition of myogenesis by deoxycytidine

The mechanism by which 5-bromodeoxyuridine (BrdU) inhibits cell differentiation is unresolved. The ability of deoxycytidine to reverse the inhibition of myogenesis produced by BrdU has been cited as evidence that the inhibition is not a direct result of the incorporation of BrdU into cellular DNA. In contrast to previous work, the present study demonstrates a direct correlation between the effects of deoxycytidine on myogenic cells and a reduction in the substitution of BrdU for thymidine in the DNA. Furthermore, the reversal occurs at the same degree of BrdU substitution (20–30%) as is required to inhibit myogenesis when cells are grown in BrdU alone or with deoxycytidine in a medium that prevents the conversion of deoxycytidine to thymidine. The effects of deoxycytidine thus do not support a mechanism of action of BrdU in myogenic cells independent of its effects on DNA.     

Lymphocyte suppressor activity in atopic eczema

Previous studies have shown that patients with atopic eczema have depressed cell-mediated immunity. Whether this defect can be attributed to abnormal suppressor cell activity or to the presence of mediators of the allergic response has not been studied before. Lymphocyte transformation was found to be enhanced in patients with mild eczema and markedly depressed in patients with severe eczema, when compared with normal controls. Pre-incubation of cultures for 48 hr without mitogen prior to transformation studies restored normal lymphocyte thymidine uptake in cells from severe atopics, suggesting a labile suppressor cell population, or a labile suppressor substance. Since mononuclear cell supernatants from patients with severe eczema failed to suppress lymphocyte transformation more than supernatants from normals, it is unlikely that the depressed lymphocyte function seen in severe eczema is due to an abnormal suppressor cell population. The possibility that mediators of the allergic response may be acting as a labile suppressor substance was evaluated by adding various concentrations of histamine, cyclic-AMP, or prostaglandin E₁ to lymphocytes undergoing mitogenesis. Histamine enhanced thymidine incorporation at low concentrations and depressed uptake at high concentrations; cyclic-AMP and prostaglandin E₁ have similar effects on transformation. It is possible that the enhancement of transformation seen in mild eczema and the depression of this response in severe eczema may be related to the concentrations or degree of allergic mediator release.     

Effects of dialyzable leukocyte extracts (DLEs) with transfer factor activity on leukocyte migration in vitro. III. Characterization of the antigen-independent migration inhibition factor in DLEs as a neutrophil immobilizing factor.

In earlier evaluations of the agarose LMI assay as an in vitro test for studying the nature and mechanism of action of TF, we reported the existence of a component in human DLEs which caused noncytotoxic inhibition of the random migration of human PMNs. The LMI was not dependent on the stimulation of viable mononuclear leukocytes by antigen or mitogen to effect the release of mediators of cellular immunity such as LIF; rather, the LMI was promoted by the direct action of a preexisting component in DLEs on PMNs. We now present evidence that this "antigen-independent" LMI activity in DLE'S is similar to a NIF shown previously by Goetzl and co-workers to be present in acid extracts of leukocytes and to be released by phagocytosing PMNs. The comparison is drawn from several parameters: (1) cellular origin, (2) molecular weight, (3) target cell, (4) susceptibility to inactivation by heating or by incubation with pronase, trypsin, or chymotrypsin, and (5) ability to cause noncytotoxic inhibition of random migration or chemotaxis of PMNs.     

Benoxaprofen activation of suppressor activity in mononuclear leucocytes by a pro-oxidative mechanismin vitro

The effects of benoxaprofen on spontaneous and concanavalin A-induced suppressor activity in human mononuclear leucocytes (MNL) were assessedin vitro. The drug was used at a fixed concentration of 10^–4 M (30 g/ml) in these studies. Benoxaprofen-treated MNL suppressed the responsiveness of untreated autologous MNL to the mitogen phytohaemagglutinin and potentiated the induction of suppressor activity in MNL by concanavalin A. Benoxaprofen at the same concentration increased MNL oxidative metabolism measured by chemiluminescence. Inclusion of the anti-oxidants ascorbate or cysteine (1×10^–3 M) in the assay system or depletion of adherent cells from MNL populations was associated with the elimination of both benoxaprofen-mediated suppression and increased MNL oxidative metabolism. Benoxaprofenper se was not an oxidizing agent nor did the drug possess peroxidase-like properties. These findings show that benoxaprofen induces suppressor activity in MNL by a pro-oxidative mechanism dependent upon intact cellular oxidative metabolism. Induction of suppressor activity in MNL by pro-oxidative drugs may be an important anti-inflammatory mechanism.     

Mechanisms of inhibition of mononuclear cell activation by the iron-chelating agent desferrioxamine.

Iron-withholding by the chelating agent desferrioxamine abrogates the proliferative response of human peripheral blood mononuclear cells (PBMC) to phytohaemagglutinin (PHA). The present study investigated whether desferrioxamine operates late in the activation process or, as recently suggested, at an early stage, by inhibiting the appearance of the interleukin-2 (IL-2) receptor. Human PBMC were stimulated with PHA (10 micrograms/ml) and [3H]thymidine ([3H]TdR) incorporation determined after 66 hr of culture. Greater than 90% inhibition was achieved by concentrations of desferrioxamine as low as 5 mumol/l present throughout culture, while IL-2 receptor expression (anti-Tac), analysed by FACS, was maintained at up to 75% of control levels. 300 mumol/l desferrioxamine present throughout culture abrogated [3H]TdR incorporation and additionally suppressed IL-2 receptor to 10-15% of control levels. In contrast, the same high dose of desferrioxamine when added for 2 hr to cells previously cultured for 66 hr produced 80% inhibition of [3H]TdR incorporation but failed to inhibit expression of the IL-2 receptor. Desferrioxamine rapidly achieved equilibrium across the cell membrane (within 60 min) and chelated 59Fe delivered to activated cells by the transferrin endocytic cycle. These results indicate that desferrioxamine can inhibit T-cell activation either early or late in the process by chelating iron and independently of an effect on the IL-2 receptor. In support of a dual effect of the drug is the finding that at 50 mumol/l, desferrioxamine-enhanced expression of the transferrin receptor occurred, an adaptive response made to intracellular iron depletion, while IL-2 receptor expression was inhibited.     

Effects of immunoglobulin G from patients with systemic lupus erythematosus on human B cell function

We examined the effect of systemic lupus erythematous (SLE) sera and Ig fractions on IgG and IgM release by cultured normal peripheral blood mononuclear cells (PBMC) when these cells were preincubated with serum dilutions or Ig fractions. Increases in both IgM and IgG (P less than 0.001 and less than 0.01) in cultured cell supernatants were recorded when PBMC were preincubated with SLE serum dilutions. IgG but not IgM from SLE was found to stimulate PBMC to release IgG (P less than 0.01). Similar results were obtained when SLE IgG was preincubated with adherent cell depleted cells (ADC) or isolated normal B cell fractions. When normal PBMC were preincubated with SLE serum or IgG and subsequently stimulated with pokeweed mitogen (PWM), a relatively blunted IgG release was observed (P less than 0.05); however, IgM release was significantly increased (P less than 0.001). This effect was not observed when PBMC were preincubated with SLE IgM, normal serum dilutions, or normal Ig fractions. Relative blunting of PWM response after PBMC were preincubated with SLE IgG was not reversed in PBMC depleted of adherent cells, OKT8+, or OKT9+ cells. Depletion of PBMC of LeuM1 cells increased IgG release in response to PWM when cells had been preincubated with SLE IgG. SLE serum or Ig fractions did not induce B cell growth factor release by T cells. SLE IgG appeared to act directly on B cell enriched populations to release IgG; this was not associated with significant increase in thymidine uptake, or apparent lysis of cells.     

Carbon monoxide reversibly alters iron homeostasis and respiratory epithelial cell function

The dissociation of iron from heme is a major factor in iron metabolism and the cellular concentrations of the metal correlate with heme degradation. We tested the hypotheses that (1) exposure to a product of heme catabolism, carbon monoxide (CO), alters iron homeostasis in the lung and in cultured respiratory epithelial cells; (2) this response includes both decreased uptake and increased release of cell metal; and (3) the effects of CO on cell function track changes in metal homeostasis. In rats exposed to 50 ppm CO for 24 hours, non-heme iron concentrations decreased in the lung and increased in the liver. In respiratory epithelial cells cultured at air-liquid interface, CO exposure decreased cell non-heme iron and ferritin concentrations within 2 hours and the effect was fully reversible. CO significantly depressed iron uptake by epithelial cells, despite increased expression of divalent metal transporter-1, while iron release was elevated. The loss of non-heme iron after CO reduced cellular oxidative stress, blocked the release of the proinflammatory mediator (interleukin-8), and interfered with cell cycle protein expression. We conclude that CO reduces the iron content of the lung through both the metal uptake and release mechanisms. This loss of cellular iron after CO is in line with certain biological effects of the gas that have been implicated in the protection of cell viability.