Thomas Hoffman

Release of arachidonic acid metabolites by human monocytes or lymphocytes: Effect of treatment with interferon on stimulation by phorbol ester or calcium lonophore

The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids was studied in human peripheral blood monocytes obtained by counter-current centrifugal elutriation. Monocytes prelabeled for 4 hr with [3H]arachidonic acid (AA) released label into the surrounding medium in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore (A23187). High-performance liquid chromatography of monocyte supernatants demonstrated that labeled compounds included those which eluted with authentic standards for thromboxane B2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 6-keto-prostaglandin F alpha, prostaglandin E2, and 15-hydroxyeicosatetraenoic acid (HETE). 5-HETE and leukotriene B4 (LtB4) were detected only in response to ionophore treatment. Highly purified lymphocytes did not convert AA to autocoids, despite the release of free arachidonate in response to either stimulus. Pretreatment of monocytes with recombinant human interferon (IFN)-gamma or IFN-alpha for 18 hr resulted in enhanced release of labeled arachidonic acid and increased conversion to autocoids after TPA or ionophore stimulation. Absolute amounts of prostaglandin E2 produced in response to TPA or ionophore treatment were increased as well. These results demonstrate the autocoid profile released by stimulated human monocytes and illustrate the effects of IFN treatment on the production of lipoxygenase metabolites of arachidonic acid as well as cyclooxygenase products.

Characterization of purified cryopreserved human monocyte function in assays of superoxide production, accessory cell function, chemotaxis, and in fluorescent cell sorter analysis.

The ability to use highly purified cryopreserved human monocytes in various in vitro assays has a number of practical and theoretical advantages, including convenience and the potential for enhanced reproducibility. Large numbers (up to 1 × 109) human monocytes can be isolated from a single donor in a purified suspension state, by a combination of leukapheresis and counter‐ current centrifugal elutriation (CCE) technologies. Following short‐ and long‐ term periods of cryopreservation, the viability and phagocytic function of these CCE‐purified monocytes was unimpaired. Cryopreserved monocytes were similar to fresh cells in their ability to release superoxide anion (O2), although unstimulated and stimulated Of release values tended to increase slightly following weeks to months of cryopreservation. In contrast, even short‐term cryopreservation diminished both the random migration and chemotactic responses of human monocytes; however, cryopreserved monocytes could be employed in this assay provided the calculated chemotactic ratio (chemotactic migration/spontaneous migration) was used. Cryopreserved monocytes demonstrate 70% of fresh monocyte accessory cell function in pokeweed mitogen‐ induced lymphocyte proliferation assays. When the binding of OKT3, OKM1, anti‐DR, and fluoresceinated pokeweed mitogen to monocytes was analyzed in the fluorescence activator cell sorter (FACS), cryopreserved and fresh monocytes displayed a similar pattern of membrane reactivity.

Functional Consequences of Phospholipase A2 Activation in Human Monocytes

Human monocytes release arachidonic acid upon stimulation with a variety of soluble or particulate agents. These include: phorbol esters (i.e., 12-O-tetradecanoate phorbol-13-acetate, TPA), calcium ionophores (ionomycin), serum-treated zymosan (STZ) concanavalin A (Con A), and, to a minor degree, lipopolysaccharides (LPS). Protein Kinase C activation or increased intracellular Ca2+ are common features of the actions of most, if not all, of these stimuli. Prevention of PKC activation by the use of staurosporine or chelation of extracellular calcium by EGTA selectively impaired AA release, indicating that PLA2 may be regulated by either pathway concurrently. The generation of inositol phosphates and diacylglycerol by the action of phospholipase C, notably upon interaction with opsonized particles during phagocytosis, apparently constitutes the physiological correlate of stimulation via these agents. Release of arachidonic acid by the action of PLA2 or other phospholipid hydrolyzing enzymes leads directly to the formation of cyclooxygenase products. In the presence of markedly elevated calcium concentrations, 5-lipoxygenase (LO) is activated as well, leading to the formation and release of leukotrienes. Agents which stimulate AA release also initiate other monocyte functions, including generation of reactive oxygen intermediates and lymphokine release. This observation makes it tempting to implicate PLA2 activation in many aspects of monocyte physiology. However, no correlation with PLA2 activation and either superoxide or lymphokine release was found when multiple stimuli, including TPA, ionomycin, serum-treated zymosan, concanavalin A, or LPS, were compared simultaneously. Instead, our results indicate that PLA2 activation is regulated by the same mechanisms, including PKC activation and increased Ca2+, as are other enzymes which determine expression of monocyte function. Phospholipase A2 (PLA2) hydrolyzes fatty acid from the sn-2 position of a wide variety of phospholipids. Substrates for this (these) enzyme(s) include species which contain a variety of polar head groups (choline, serine, ethanolamine, etc.) and some phospholipids with either linkages in sn-1. In many cell types, including human monocytes, phospholipase A2 commonly acts on substrates containing arachidonic acid (AA). The liberation of free arachidonate is a first step in the metabolism of prostaglandins, hydroxyeicosatetraeinoic acids, (HETES), and leukotrienes (Lts). Monocytes and macrophages have been shown to be rich sources of arachidonate and its metabolites. Some biologic properties of monocytes, notably their role as immunomodulating cells, have been attributed to eicosanoid production and release. Accordingly, much of the interest regarding PLA2 in human monocytes centers on this aspect of their function.(ABSTRACT TRUNCATED AT 400 WORDS)

Inflammatory mediator release from human monocytes via immobilized Fc receptors. Its potential role in adverse reactions to systemic monoclonal antibody therapy.

&NA; Human monocytes released superoxide anion, IL-1, and TNF subsequent to binding of their Fc receptor I to murine IgG2a or rabbit IgG. Fc receptor II binding to murine IgG2b or IgG1 had similar consequences. Immobilized murine monoclonal antibodies, IgG2a anti-CD3 (OKT3) or IgG1 anti-CD44 also induced superoxide anion and monokine production. Monocytes bound OKT3 via FcRI and responded to immobilized OKT3 by inflammatory mediator release in the absence of T cells. These results suggest that direct interaction of immunoglobulins with monocytes via FcR may represent an important phase of the pathophysiology of adverse reactions to systemic monoclonal antibodies.

Effects of serine protease inhibitor, tame, on IL-1β in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1β (mIL-1β) but did not result in release of precursor IL-1β (pIL-1β). In contrast, the presence of a serine protease inhibitor, Nα-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1β as well. These effects were initially discerned from observations that the fraction of the total IL-1β produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1β. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1β synthesis, predominantly affecting 31-kDa pIL-1β, and a dose-dependent inhibition of TNF-α production. Parallel examination of lactate dehydrogenase (LDH) release indicated thatpIL-1β release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1β in situ but are probably not mechanistically related to either maturation of the IL-1β molecule or signaling of IL-1β release. IL-1β release appears to be dependent on the amount of total IL-1β synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1β.

T lymphocyte aggregation with immobilized anti-TCR-antibodies is dependent upon energy and microfilament assembly

An assay has been developed to quantitate the binding of beads coated with anti-T cell receptor (TCR) monoclonal antibodies (MoAb) to T lymphocytes. The Ab used were a hamster MoAb, 145.2C11 (2C11), directed against the epsilon chain of the CD3 complex of the murine TCR, and a murine MoAb, F23.1, directed against the V beta 8-encoded determinant of the alpha/beta heterodimer of the TCR. Ab were adsorbed onto polystyrene beads and the beads labeled with [125I]bovine serum albumin [( 125I]BSA). The labeled, Ab-coated beads were mixed at 4 degrees C with murine, cloned T-helper (Th) cells and contact between beads and cells was promoted by centrifugation. The mixtures were incubated at 37 degrees C for 10-20 min, and unbound beads were separated from cell-bound beads by Percoll gradient centrifugation. Beads coated with anti-TCR Ab formed stable conjugates with Th cells; an average of 6-10 2C11 Ab-coated beads/cell, or 10-15 F23.1 Ab-coated beads/cell was measured under optimal conditions. Beads coated with control Ab (hamster or mouse IgG) did not appreciably bind to the cells. Conjugation with 2C11 Ab-coated beads could be prevented by coating the cells with soluble 2C11 Ab, but not with soluble F23.1 Ab. Blocking the CD3 epsilon chain with soluble 2C11 Ab also reduced conjugate formation with F23.1 Ab-coated beads, suggesting a steric hindrance phenomenon. The extent of conjugation depended on the density of immobilized Ab. Maximum conjugation was observed when 100 micrograms of 2C11 Ab was used to coat 10(6) beads; higher Ab amounts did not further increase binding. Increasing the bead to cell ratio in the mixture increased binding, reaching optimal binding at 300:1, irrespectively of the amount of Ab adsorbed onto the beads. Stable binding of anti-TCR Ab-coated beads to T cells was temperature and energy dependent. It was prevented when glucose was removed from the medium and the glycolysis inhibitor, 2-deoxy-D-glucose was added, or when cells were treated with sodium azide. Conjugate formation was prevented by pretreatment of the cells with cytochalasins, indicating that microfilament assembly was essential. Microtubules were not involved, as vinca alkaloids were without effect. This novel assay system provides a simple means of studying aspects of TCR function including its physical and metabolic regulation.

Suppression of B cell immunity to DNP by serotherapy with monoclonal anti-DNP antibodies. Effect of the treatment schedule on the magnitude of specific suppression and its duration.

The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) monoclonal antibodies on the humoral immune response of BALB/c mice was examined. One-time administration of a cocktail of two anti-DNP monoclonals resulted in suppression of the IgM anti-DNP response for one week after challenge but not longer. Maximal suppression of anti-DNP IgM plaque-forming cells was achieved by administration of antibody 1-2 weeks before challenge with DNP. Maximal suppression of serum IgM antibody was seen by administration of antibody 2-3 weeks before challenge with antigen. Following one-time administration of suppressive monoclonal antibody, the serum IgG antibody response to DNP was suppressed beginning 2 weeks after immunization and remained so for up to 241 days despite continual booster injections of antigen. Although most effective suppression of the humoral anti-DNP response was seen in animals receiving their single dose of suppressive antibody 2 weeks before first exposure to antigen, suppression of the IgG response was evident at all intervals examined up to 232 days in mice given monoclonal antibody between 0.1 day and 30 days before antigen, but not at earlier times. These findings suggest that regulatory networks, rather than the masking of antigenic determinants by passively administered antibody, play a role in antibody-mediated immunoregulation. They may be of use in designing strategies for optimizing immunosuppression protocols in clinical studies.