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Interleukin-4 (IL-4) induced a time- and dose-dependent production of leukotriene B4 (LTB4) by human resting monocytes indicating that IL-4 induced the activation of the 5-lipoxygenase pathway in resting human monocytes. Maximal effect was observed in the presence of 10 ng/ml IL-4, and in kinetics experiments LTB4 production plateaued 40 min after the onset of stimulation. When stimulated for 48 hr with IL-4, resting human monocytes expressed and released the low-affinity receptor for IgE (CD23) and were partially inhibited in the presence of a highly non-redox 5-lipoxygenase inhibitor (BW B70C), suggesting that the production of LTB4 partially contributed to the IL-4-induced CD23 expression and release. This hypothesis was strengthened by the fact that exogenous LTB4 (10 nM) was found to increase the effect of a suboptimal dose of IL-4 (1 ng/ml). In addition to these phenotypical changes, IL-4 primed the phorbol-12-myristate-13-acetate (PMA)-induced luminol-dependent chemiluminescence response (LDCL) by normal human monocytes, this priming effect being abrogated in the presence of BW B70C. Taken together, these data indicated that IL-4 induced the production of LTB4 by activation of the 5-lipoxygenase pathway in human monocytes, and that the activation of this pathway could upregulate the expression and release of CD23 and the respiratory burst of these cells.

The capacity of human peripheral blood mononuclear cells and monocytes to generate nitrites, spontaneously or in response to Interleukin-4 was evaluated in vitro. Peripheral blood mononuclear cells and monocytes were found to release significant amounts of nitrites after 8 to 12 days in culture. This spontaneous production of nitrites was inhibited in the presence of 1 mM N G monomethyl- l -arginine, suggesting that this process was dependent upon the l -arginine metabolism. The present data also indicated that addition of Interleukin-4 generally resulted in an increased nitrite production, that was potentiated by IFN-γ, inactive alone. The response of human monocytes to Interleukin-4 was more heterogenous than that observed with unfractionated peripheral blood mononuclear cells. These results suggest that cell/cell interactions could play an important role in the activation of the nitric oxide synthase pathway in human.

Resting human blood monocytes from some donors were found to produce a small amount of 3'-5' guanine cyclic monophosphate (cGMP) in response to interleukin 4 (IL-4). A much higher response was observed when monocytes were preincubated with interferon (IFN-gamma), which alone was ineffective. Preincubation of monocytes with IL-4 led, in contrast, to their subsequent incapacity to generate cGMP in response to IL-4. The accumulation of cGMP induced by IL-4 in IFN-gamma preincubated monocytes was dose-dependent and peaked about 15 min after its addition. It was inhibited in the presence of NG-mono-methyl-L-arginine (L-NMMA), an inhibitor of the nitric oxide synthase pathway. This suppressive effect of L-NMMA was reverted by an excess of L- but not of D-arginine. Accumulation of cGMP was significantly reduced by addition of soluble guanylyl cyclase inhibitors, such as LY83583 [correction of LY83853] and methylene blue, but was not impaired in the presence of EGTA, suggesting that the pathway involved is calcium independent. In addition, IL-4 induced an increased secretion of nitrite by monocytes, that was potentiated by IFN-gamma and inhibited by L-NMMA. Taken together, these results suggest that the sequential exposure of monocytes to IFN-gamma and IL-4 elicits the release of NO from L-arginine, which in turn is capable to stimulate soluble guanylyl cyclase.

Interferon-gamma activates both in vitro and in vivo macrophage functions. Injection of rat recombinant interferon-gamma (rR-IFN-gamma) induced the expression of interleukin-2 receptors (IL-2R) by peritoneal macrophages from normal BALB/c and MRL-+/+ mice. Moreover, rR-IFN-gamma stimulated in a dose-dependent manner the oxidative burst of cells as revealed by luminol-dependent chemiluminescene (LDCL). Resident peritoneal macrophages from MRL-lpr/lpr (mice that develop a systemic lupus-like syndrome) showed a higher PMA-triggered LDCL response. This enhanced activity was accompanied by an increase in IL-2R expression (30% vs. less than 1%). The "activated" macrophages from rR-IFN-gamma-treated normal mice as well as MRL-lpr/lpr mice did not respond to the addition of recombinant interleukin-2 (rHu-IL-2) by an increase in LDCL. However, rHu-IL-2 triggering became efficient when cells enriched in IL-2R-bearing macrophages were preincubated overnight with rHu-IL-2R. This response may point out a functional role for IL-2R and provide a role for IL-2 in certain macrophage functions.

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.

Levels of plasma lactoferrin are decreased in HIV-1-infected patients in relation to the progression of the disease. Plasma lactoferrin concentrations were determined using a specific and sensitive enzyme immunoassay. 97 plasma were studied (22 asymptomatic, 45 symptomatic patients compared to 30 healthy controls) and the results showed a highly significant decrease (p0.001) of the level of lactoferrin in HIV-1-infected patients (respectively 2.79 +/- 1.2 and 0.68 +/- 0.22 micrograms/ml) compared to controls (4.37 +/- 0.83 micrograms/ml). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, the experiments were reproduced in neutropenic patients who represent 10% of recruitment (6 among 45 symptomatic patients). The plasma from neutropenic symptomatic patients (neutrophilsor = 1,300/mm3) showed their mean lactoferrin level at 0.36 micrograms/ml still far above the normal values. In view of the different reported biological effects of lactoferrin that are of great importance in the non-specific defences, the real biological place of the lack of such a molecule could be one important component of the multifactorial nature of HIV-1 infection.

Human monocytes, preincubated with IFN-γ respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 – 3 min) and cAMP (20 – 25 min) in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1) IL-4 may stimulate different NOS isoforms in resting and IFN-γ activated monocytes, and (2) cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca2+]iin these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.

Resting normal human monocytes were found to produce small amounts of cGMP in response to IL-4. This production was inhibited in the presence of LNMMA suggesting an association with activation of the NO synthase (NOS) pathway. In addition, this cGMP generation was abrogated in the presence of either a Ca2+ chelator, EGTA, or a calcium/calmodulin inhibitor, W7, suggesting that IL-4 stimulates the constitutive NOS (cNOS). An enhanced response was observed when monocytes were preincubated with IFN-gamma and whether the cGMP accumulation was still abrogated in the presence of LNMMA it was not affected by either EGTA or W7 suggesting, in that case, the activation of an inducible NOS (iNOS). Taken together these data suggest that IL-4 could stimulate a cNOS in resting and an iNOS in the IFN-gamma-treated human monocytes, indicating that the generation of NO is highly dependent on the maturation state of these cells.

An in vitro study was performed in order to assess a possible regulatory role of nitric oxide (NO), a short-lived biologic mediator that displays immunoregulatory properties, in the IL-4-driven synthesis of IgE by normal human peripheral blood mononuclear cells (PBMC). In addition to induce IgE production, IL-4 was found to elicit nitrite (NO2-) release by PBMC. A marked correlation was observed between IgE secretion and nitrite release by PBMC stimulated with an optimal concentration of IL-4. The IL-4-dependent IgE production was significantly reduced (p0.001) in the presence of N omega-monomethyl-L-arginine (LNMMA), an inhibitor of the NO-synthase pathway; this inhibition was partially reverted with an excess of L-arginine. Addition to PBMC cultures of the chemical NO donor Sin-1, inactive alone, was found to result, depending on the concentration of IL-4, in either potentiation (suboptimal concentration of IL-4, 10 ng/ml) or inhibition (optimal concentration of IL-4, 50 ng/ml) of IgE synthesis. The potentiating effect of Sin-1 was dose dependent, with a maximal effect for 300 microM, whereas its metabolite Sin-1c was inactive. In both cases, Sin-1 markedly reduced the IL-4-induced release of the soluble form of the low affinity IgE receptor (sCD23). Together, these data strongly suggest that NO may display biphasic immunoregulatory properties on the IL-4-induced IgE production by PBMC.

Sera from 85 HIV-infected patients were tested for the presence of antilactoferrin antibodies (anti-LF Abs) by specific ELISA. Fifty-seven sera were found positive, including sera from asymptomatic (18/28, 64.3%, mean O.D.: 0.27 +/- 0.05) and symptomatic patients (39/57, 68.4%, mean O.D.: 0.82 +/- 0.15). In the control group, only one out of 26 normal donors show any reactivity (mean O.D.: 0.06 +/- 0.01). None of the tested patients had clinical evidence of vasculitis, the previous reported antilactoferrin-associated pathology and if, in both groups, a similar frequency of anti-LF Abs was found, the autoantibody level was significantly higher among the symptomatic patients (p0.01). However, correlation was found neither with polymorphonuclear cell counts nor with the level of circulating lactoferrin. The characterization and the clinical significance of the autoantibodies are under investigation.

Lf presented differential effects in the induction of normal human monocyte activation. Lf inhibits the free radical generation by normal human monocytes in response to PMA. This effect on free radical generation is linked to the nature of the divalent cationic metal that substitutes the Apo-Lf. Indeed, the substitution of Fe2+ by another cationic transition metal (Cu2+, Zn2+, Pt2+ or Au2+) modifies the anti-oxidant effect of Lf. Among the different substituted Lf the Lf saturated with Cu2+ is the more potent inhibitor of free radical generation by normal human monocytes. In addition to its anti-oxidant effect, Lf stimulates, in combination with LPS, the production of cytokines (IL-1 beta, TNF-alpha and IL-6) and of prostacyclin (PGI2) by normal human monocytes. This potentiating effect is independent of the cation that substitutes the Lf, since the Apo-Lf is able to induce such a potentiating effect. In addition, whereas Lf does not modulate the phenotype of normal human monocytes stimulated or not with IL-4 it does enhance the IL-4-induced release of the soluble form of CD23 by these cells.

3'-azido-3'-deoxythymidine (Azidothymidine or AZT) has attained wide critical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, treatment with AZT is associated with the development of severe hematopoietic toxicity. The AZT sensitivity of marrow progenitors was different with an IC 50 of 10(-8) M and 10(-6) M for respectively BFU-E and CFU-GM/GEMM. Data reported here show that recombinant human interleukin-1 alpha (IL-1 alpha), a pleiotropic cytokine, was demonstrated to be efficient to protect normal human as well as murine hematopoietic progenitors (CFU-GM, CFU-GEMM and BFU-E) from the toxic effect of AZT. The maximal effect was observed with 30 U/ml (Human cells) or 100 U/ml (murine cells) IL-1 alpha for BFU-E and CFU-GM/GEMM, with a marked effect at 1 U/ml. The results demonstrate that marrow progenitors respond differently to AZT and point out the potential efficacy of IL-1 alpha to enhance the proliferation of hematopoietic stem cells treated with growth factors (IL-3, erythropoietin) and to minimize the hematopoietic toxicity associated with AZT treatment.

Human dermal fibroblast proliferation was dependent of growth factors such as interleukin-1 (IL-1) and fibroblast growth factors (FGFs). These mediators, which induce proliferation of the cells in culture, were able to synergize when added in combination. This synergistic effect seems to be restricted to the mitogenic activity since IL-1-induced PGE2 release and interferon-beta 1 (IFN-beta 1) production by fibroblasts was inhibited in the presence of FGFs, which by themselves were unable to stimulate the production of IFN-beta 1 and the release of arachidonate metabolites from the fibroblasts even at high concentration.

paf-Acether (paf) is a phospholipid mediator of inflammation released from monocytes along with IL-1. In this study, we have examined the role of paf on IL-1 production by human monocytes. When paf from 1 nM to 5 microM, but not its precursor lyso paf, was added to monocytes in the presence of muramyl dipeptide (MDP) or LPS, a marked increase in IL-1 activity over the value with MDP alone was observed. In contrast, paf alone had minimal activity over the same dose range. Antibodies against rHu IL-1 alpha and rHu IL-1 beta neutralized the increased IL-1 activity. Interestingly, MDP that prompts monocytes to synthesize IL-1, induced the synthesis of paf, as well. Most of the paf produced remained cell-associated and always preceded IL-1 synthesis. When the paf receptor antagonist, L-652,731 was added to monocytes, it prevented the enhancement of IL-1 activity induced by exogenous paf. In contrast, L-652,731 had little effect on MDP-induced IL-1 synthesis in the absence of exogenous paf. This may indicate that there are alternative mechanisms involved in the sequences of events leading to IL-1 production. It is also conceivable that the paf receptor antagonist is not able to compete or inhibit endogenous paf as well as it does for exogenous paf. Nevertheless, exogenous paf in association with a second signal, modulates IL-1 production from human monocytes in a positive manner. This may constitute another means through which paf can modulate inflammatory and immune reactions.

Recombinant human Interleukin-1 (rhlL-1) β was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhlL-1β and then stimulated with either heat-aggregated IgG (Hagg) or N-formyhmethionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 μg/ml Hagg, 2.5 x 10-9 M FMLP) did not stimulate degranulation in the absence of rhlL-1β. However, such concentrations elicited marked degranulation from PMNs preincubated with rhlL-1β (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1β employed and on the period of incubation. In other experiments, the effect of rhlL-1β on the PMN oxidative response was determined. rhlL-1β did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1β may potentiate PMN degranulation, but not their oxidative response.

Differentiation of HL60 cells into neutrophil-like cells after exposure to dimethylsulfoxide is accompanied by an increase in intracellular pH (pHi) which results from an increased activity of the Na+/H+ antiporter at physiological pHi value, but not at acidic pHi values. The functional responses of differentiated HL60 cells to the chemotactic peptide, N-formylmethionylleucylphenylalanine (fMLP), and to phorbol myristate acetate (PMA) were studied. In differentiated cells fMLP produced a large increase in cytosolic Ca2+ levels and a small biphasic change in pHi, whereas PMA produced cellular acidification, which was potentiated by ethylisopropylamiloride and no change in [Ca2+]i. In undifferentiated HL60 cells, PMA produced the opposite effect on pHi, i.e., a cellular alkalinization. The extent of the acidification produced by PMA in differentiated HL60 cells correlated with the production of reactive oxygen species.

Two purified peptidoglycans extracted from E. coli or B. megaterium strongly stimulated the spleen lymphocytes of rabbits and of normal or nude mice. Both preparations can substitute for Mycobacteria in Freund's complete adjuvant. The peptidoglycan extracted from M. lysodeikticus by a similar procedure which lacks adjuvant activity, did not induce blast transformation. However, the monomere of the E. coli peptidoglycan was devoid of mitogenicity although it has also a marked adjuvant activity.

Purified polymorphonuclear (PMN) cells were obtained from human blood leukocytes by centrifugation on colloidal silica gradients. PMNs could be primed for PMA-triggered oxidative burst by muramyl peptide molecules (MDP) and two of its adjuvant active nonpyrogenic derivatives. The priming effect of MDP could be demonstrated after a 1-h incubation period, whereas monocytes needed an 18-h incubation to produce an enhanced response in the NBT reduction test. Only the monocyte-enriched population was able to produce IL-1 activity after muramyl peptide stimulation. Under such conditions, PMNs neither produced nor secreted IL-1-like activity, and no IL-1 inhibitor was present in the supernatant fluids. In conclusion, muramyl peptides were able to prime PMNs for oxidative burst but not to stimulate IL-1-like factor production.

N -acetylmuramyl-L-alanyl-D-isoglutamine or MDP is a synthetic immunoadjuvant capable of inducing a febrile response in the rabbit and of eliciting production of endogenous pyrogen (EP) in vivo and in vitro . It was also shown to stimulate in vitro lymphocyte activating factor (LAF) release. After conjugation to a synthetic carrier, the pyrogenic effect of MDP is strongly enhanced. Recently, adjuvant active derivatives of MDP devoid of pyrogenicity such as MDP(Gln)-O n Bu (AcMur-L-Ala-D-Gln- α - n butyl ester) have become available. In the present study, their stimulating properties on rabbit peritoneal exudate cells or human peripheral blood mononuclear cells were simultaneously evaluated by testing lymphocyte activating property and pyrogenic effect of the culture supernatant. In both types of cell cultures, whereas MDP elicited dual effects, the nonpyrogenic analog MDP(Gln)-O n Bu produced LAF without detectable pyrogenicity even when a very sensitive assay (intracerebroventricular administration) was used. In contrast, conjugated MDP was capable of inducing the production of endogenous pyrogen in rabbit cell cultures with no or little LAF release. These results argue against the possibility that a single factor is responsible for both LAF and EP activities.

A water-soluble extract of Nocardia opaca (NWSE) induced a very strong stimulation of rabbit and mouse spleen lymphocytes and a weak, but significant, increase of thymidine incorporation by human peripheral lymphocytes and monkey blood, or spleen cells. As was demonstrated by the migration inhibition factor assay, the strong mitogenic activity observed with mice spleen cells was not related to a state of natural impregnation. In the same experiments, the various lymphocytes were also incubated with M. smegmatis water-soluble adjuvant (WSA) and S. enteritidis lipopolysaccharide (LPS). No mitogenic activity was shown with WSA even when this extract was incubated with the lymphocytes of human tuberculin responders, showing that this preparation is free of tuberculoprotein contaminants. Finally, it must be noted that, contrary to what is observed in mice, LPS did not induce thymidine incorporation by monkey or rabbit spleen cells.

N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide or MDP) is the minimal structure required for substituting mycobacteria in Freund's complete adjuvant. It is an adjuvant when injected in saline, protects mice non-specifically against infection, and enhances thymidine incorporation of lymphocytes. In this report it is shown that the linking of L-Lys, L-Ala-D-Ala, L-Lys-D-Ala or L-Lys-L-Ala to MDP permits the dissociation of anti-infectious activity from adjuvant activity. The optical configuration of the added residues plays the major role in this dissociation. It can be noted that the muramyl tetrapeptide MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala mimicking the natural structure is devoid of anti-infectious activity.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti-RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

It has been previously observed that lipopolysaccharides can be detoxified by alkylation and yet retain their adjuvant activity. Our present findings confirm these results and show, moreover, that these derivatives did not lose their capacity to protect mice against lethal irradiation and lost only partially their ability to interrupt pregnancy or to induce blast transformation of murine B-lymphocytes. However, in contrast with lipopolysaccharides, these alkylated preparations did not enhance the nonspecific resistance of mice to a bacterial infection. The various bilogical functions of endotoxins can therefore be separated and are not uniformly related to their toxicity.

Treatment of rabbit spleen cells with a water soluble mitogen from Nocardia opaca (NWSM) resulted in the appearance of 30% of cells with the ultrastructural characteristics of plasmocytes and in enhanced thymidine uptake. The extent of incorporation was not diminished by prior complement mediated killing of thymus derived cells with a goat antibody, directed against rabbit thymus lymphocyte antigen (RTLA). The exposure of rabbit spleen cells to NWSM resulted in a polyclonal antibody response. It was concluded that NWSM is a potent mitogen for bursa equivalent lymphoid cells of the rabbit.

A fall in plasma iron level and an increase in copper level were observed in rabbits subsequent with the febrile response induced by an intravenous administration of muramyl dipeptide, AcMur-L-Ala-D-isoGln (MDP). The pyrogenic activity of MDP was due partly to the induction of circulating endogenous pyrogen (EP). EP produced in vitro by activated macrophages also elicited changes in iron and copper levels in rabbits. Nonpyrogenic MDP derivatives murabutide [MDP(Gln)-OnBu] and the stereoisomer of MDP [MDP(D,D)] did not cause any change in blood metal levels. Another adjuvant and nonpyrogenic analogue, murametide [MDP(Gln)-OMe], elicited hypoferremia and hypercupremia. Murametide, which has been previously shown to induce secretion of circulating EP but prevents in vivo fever response, was unable to prevent an EP-induced effect on plasma metal concentrations. Injection of supernatant fluids of macrophages incubated with these different glycopeptides showed that only compounds able to induce EP release were capable of evoking hypoferremia and hypercupremia. The EP-containing fluid was 10-fold more active on change in temperature and in plasma metal levels when it was given intracerebroventricularly compared with intravenously. In contrast, a pyrogenic dose of MDP that can act directly on the central thermoregulatory structures did not modify iron and copper levels when it was injected intracerebroventricularly.

Muramyl dipeptide (MDP) and adjuvant-active derivatives were confirmed as unable directly to induce differentiation of mouse myeloid leukemia M1 cells. They were, however, found effective in stimulating either rabbit macrophages or human blood monocytes to produce differentiation-stimulating activity (D factor). The various conditioned media (CM) thus obtained were able to induce differentiation of the myeloblastic M1 cell line as indicated by the appearance of Fc receptors and inhibition of cell proliferation. Among the synthetic glycopeptides inducing the production of D factor, murabutide (MDP[Gln]-OnBu) was as effective as MDP, although it did not stimulate monocytes to simultaneously release endogenous pyrogen. The absence of pyrogenicity in murabutide CM was attested by IV or intracerebroventricular administration to rabbits. However, in the same CM, LAF(IL1) activity estimated by potentiation of the in vitro proliferative response to phytohemagglutinin of mouse thymocytes was usually higher than that induced by MDP.

Two immunologically different mitogens have been purified from culture fluid of growingStreptococcus pyogenes (group A). These substances were shown to be highly active on human and rabbit lymphocytes from peripheral blood, thymus and spleen, and less active on AKR mice thymocytes and splenocytes as well as on Nude mice splenocytes. These substances therefore appear as T-cell mitogens. Their effect on B cells remains to be investigated.

Peritoneal macrophages harvested from animals undergoing an acute non-immunological inflammatory reaction induced by an injection of calcium pyrophosphate (CaPP) or dextran into the pleural cavity showed an enhanced level of production of a lymphocyte activating factor (LAF) like material. On the other hand, macrophages harvested from the site of inflammation did not show enhanced production of this activity when compared to macrophages derived from the normal controls. This enhanced activity was observed for up to two/three days after initiation of the inflammatory reaction and was obtained in both the presence and absence of lipopolysaccharide (LPS) during vitro cultivation. Thus these results demonstrate that the acute inflammatory reaction, initiated by a non-antigenic stimulus (CaPP), is able to stimulate macrophages remote from the inflammatory site to produce a factor which behaves like LAF in a standard LAF assay.

Five Monoclonal Antibodies (MAbs) coded respectively #111, 122, 206, 209 and 609 were produced against human recombinant Interleukin-1 beta (rIL-1 beta, amino acids 117-269). Four of these MAbs (#111, 122, 206 and 609) have been identified able to inhibit the biological activity of recombinant and natural Interleukin-1 beta. Competition studies suggested that three non-overlapping epitopes of the molecule were recognized by the MAbs. MAbs #609 and 206 have been selected on the basis of high affinity and used together in a two-site ELISA to detect IL-1 beta. The sensitivity of this ELISA was 1 pg/well (10-20 pg/ml). The assay did not detect rHuIL-1 alpha, rHuIL-2, rHuTNF-alpha, rHuIFN-gamma, rHuIL-6, and natural a- and b-FGF. The immunoassay was then compared to current assay such as co-mitogenic effect on murine thymocytes for detection of IL-1 beta in the intra- and extracellular compartments of IFN-gamma and/or LPS-stimulated human blood monocytes. We confirm that IFN-gamma potentiates IL-1 beta secretion in response to LPS (even with high dose 1 microgram/ml) but not the intracellular precursor of IL-1 beta. This immunoassay could be used also to detect IL-1 beta in plasma, sera and synovial fluids.

The Freeman polysaccharide - the hapten of lipopolysaccharide - induced a blast transformation of mouse and rabbit spleen lymphocytes. The magnitude of the response in rabbit was quite similar to that obtained by lipopolysaccharide whereas in mice it was weaker. The stimulation of lymphocytes of both species did not require the presence of macrophages. Similar results were obtained with four LPS and their correspondent haptens prepared from S. typhi-murium, S. abortus-equi, S. johannesburg R+ and R-.

Lipopolysaccharide (LPS) is a well known interleukin-1 inducer. Polymyxin B sulphate (PMB) is a cyclic antibiotic which neutralizes different biological properties of LPS. Under the conditions used in our laboratory, we were able to show that PMB alone could stimulate human monocytes to produce and release interleukin-1 activity, and that PMB was unable to inhibit the production of interleukin-1 by human monocytes stimulated with LPS. On the contrary, a synergistic effect was obtained which was not observed with another stimulant such as muramyl dipeptide.

In vitro and in vivo responses to lipopolysaccharide (LPS) and various other bacterial immunostimulants were compared in c3H/He low-responder mice. The principal findings were as follows. (i) Their splenic lymphocytes were stimulated by various gram-negative mitogens such as an Escherichia coli peptidoglycan, a detoxified derivative of LPS, and even endotoxins extracted by trichloroacetic acid that are known to contain protein; spleen cells of these mice were also transformed by two other B-cell mitogens extracted from acid-fast organisms. (ii) Their macrophages were refractory to LPS and weakly responsive to a mycobacterial prepartion. (iii) LPS failed to elicit nonspecific resistance in these mice against Klebsiella pneumoniae infection. (iv) Endotoxin extracted by trichloroacetic acid and a mycobacterial preparation that could increase nonspecific resistance to infection in other strains did not protect C3H/He mice against a challenge by K. pneumoniae, although both prepartions could evoke nonspecific responses of B cells in this low-responder subline.

In vitro and in vivo host reponses to lipopolysaccharide (LPS) and various immunostimulants were compared in C3h low-and highresponder mice, their F1, F2 and backcross hybrids. In contrast to the responsiveness of a congeneic high responder subline, LPS is unable to stimulate thymidine incorporation by splenocytes of the low-responder subline and to protect these mice against an unrelated bacterial infection, Moreover, although endotoxin extracted by trichloroacetic acid was mitogenic in these low-responder mice, it was still unable to increase their resistance against an infectious challenge; in addition, the LD50 of both endotoxin preparations was about 3,500 fold greater than in the high-responder mice. For this latter assay, mice were adrenaloctomized since it is well established that mouse's susceptibility to LPS is dramatically enhanced in the absence of glucocorticoids. Inheritance of susceptibility to lethal effect of endotoxin and of LPS-induced non-specific resistance to infection was also tested in both sublines, their hybrids and backcross progeny. The present findings are consistent with the hypothesis that these LPS host-responses may be determined by a single autosomal dominant gene.

Lipopolysaccharides (LPS) from gram-negative bacteria have been found to be strong mitogens for mouse spleen lymphocytes (1) but induce only a weak stimulation of human blood (1, 2), guinea pig (3) and rabbit lymphocytes (2, 4, 5). In chicken the LPS failed to activate bursa cells (B1) but transformed a small proportion of bursa-derived cells (B2) compared to the strong stimulation of these populations by anti-chicken Ig serum (6).

The capacity of MDP to stimulate LAF(IL 1) production by adherent peritoneal cells (APC) from different strains of mice was examined. It was observed that MDP could stimulate thioglycollate-induced APC from DBA/2, C57B1/6, and CBA/CA mice, but not from C3H mice. Resident APC from DBA/2 or C3H/HeJ mice were also responders and nonresponders, respectively, to MDP for LAF(IL 1) production, the positive effect of MDP being more marked on DBA/2 cells in the presence of indomethacin. Because a LPS high-responder C3H substrain (C3HeB/Fe) did not respond to MDP, it was concluded that the strain dependence with respect to the induction of LAF(IL 1) seen with both MDP and LPS was not linked. Moreover, the unresponsiveness of C3H mice to MDP was not linked to their MHC haplotype, because a second H-2k strain (CBA/CA) was a good responder to MDP stimulation.