Covalent disulfide binding of human IL-1 beta to alpha 2-macroglobulin: inhibition by D-penicillamine.

Secreted human IL-1 beta is known to have two free SH groups due to unpaired cysteines (positions 8 and 71). Alpha 2-Macroglobulin (alpha 2-M) has internal thioester bonds between cysteine and glutamate residues. Free SH groups may be generated at these alpha 2M residues through the action of proteinases, amines such as methylamine, or at a slow rate, by H2O ("aging" of alpha 2M). Thus, the possibility that IL-1 beta forms a disulfide bond with alpha 2M was investigated. 125I-labeled human rIL-1 beta (15 kDa) was incubated with fresh normal human serum or with purified alpha 2M, treated or not with methylamine. The mixtures were submitted to nondenaturing and denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. IL-1 beta bound to commercially purified "aged" alpha 2M and to alpha 2M in methylamine-treated serum but not to native serum alpha 2M. It did not bind detectably to any other serum proteins. The addition of D-penicillamine (D-pen) during the reaction of [125I]rIL-1 beta with serum or purified alpha 2M blocked the covalent binding of rIL-1 beta to alpha 2M. [125I]rIL-1 beta was removed from alpha 2M by 2-mercaptoethanol in SDS. Thus, disulfide bonds were formed between the free SH groups on [125I]rIL-1 beta and those resulting from the cleavage of the internal thioester bonds of alpha 2M. "Cold" rIL-1 beta and a Cys71----Ser71 rIL-1 beta mutant effectively competed with [125I]rIL.1 beta for binding sites on alpha 2M. When complexes of rIL-1 beta or the mutant rIL-1 beta and alpha 2M were subjected to nonreducing SDS-PAGE and subsequent Western blot analysis, the rIL-1 beta molecules were found to be present in the alpha 2M bands in a dose-dependent manner. rIL-1 beta attached to alpha 2M in the presence or absence of D-pen showed similar biological activity in the mouse thymocyte-assay. Thus, rIL-1 beta attached noncovalently to alpha 2M is biologically active. The lack of inhibition of rIL-1 beta activity by binding to methylamine-treated alpha 2M in the absence of D-pen suggests, but does not prove, that the covalently bound rIL-1 beta is also active. We concluded that human rIL-1 beta binds to alpha 2M through the Cys at position 8 and that D-pen inhibits this binding. We speculate that this inhibitory effect may contribute to the therapeutic benefits of D-pen in patients with rheumatoid arthritis.     

Adrenergic receptors in bovine retinal microvessels: presence of alpha 2- and beta- but not alpha 1-receptors

In bovine retinal microvessels, alpha 1, alpha 2- and beta-adrenergic receptors were characterized by binding assay, using [3H]prazosin, [3H]para-aminoclonidine and [125I]iodocyanopindolol as radioligands, respectively. The microvessels were purified from bovine eyes by differential centrifugation through a high concentration of bovine serum albumin followed by use of a glass bead filtration technique. In the preparation, specific binding sites for [3H]para-aminoclonidine and [125I]iodocyanopindolol were observed, whereas [3H]prazosin binding was not detected. The [3H]para-aminoclonidine binding sites localized to the microvessels were characterized by high affinity and saturability (KD: 173 +/- 9 pM; Bmax: 394 +/- 11 fmol/mg protein) as well as the [125I]iodocyanopindolol binding sites (KD: 20 +/- 3 pM; Bmax: 43 +/- 4 fmol/mg protein). Furthermore, the specificity of both binding sites was pharmacologically evaluated by measuring the inhibitory effects of various adrenergic reagents on binding. The existence of alpha 2- and beta-adrenergic receptors which were characterized by high affinity, saturability and stereospecificity, leads to the hypothesis that the retinal microcirculation is under neuronal control.     

TGF-beta down-regulates IL-1alpha-induced TLR2 expression in murine hepatocytes

We have previously reported that the proinflammatory cytokine interleukin (IL)-1alpha can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-beta up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1alpha at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1alpha but not TGF-beta or the combination of TGF-beta and IL-1alpha. TLR2 promoter activity and nuclear factor (NF)-kappaB activation by IL-1alpha were inhibited by TGF-beta treatment. Pretreatment with TGF-beta strongly suppressed IL-1alpha-induced TLR2 promoter activity and NF-kappaB activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1alpha up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-beta. These results suggest that TGF-beta suppresses the induction of TLR2 expression by IL-1alpha through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1alpha and TGF-beta in regulating TLR2-mediated SAA production in hepatocytes.     

Inverse relationship between epidermal growth factor induced proliferation and expression of high affinity surface epidermal growth factor receptors in rat hepatocytes

Rat hepatocytes express large numbers of high and low affinity surface membrane receptors (EGFR) for epidermal growth factor (EGF) but the roles of EGF and EGFRs in hepatocyte proliferation in vivo are unclear. F344 rat hepatocytes in primary culture proliferated maximally in response to continuous serum-free culture with 3.3 nM (20 ng/ml) EGF, as quantified by cumulative [3H]thymidine labeling index. However, serum concentrations of EGF in rats with normal livers or induced hepatocyte proliferation due to partial hepatectomy, carbon tetrachloride-induced necrosis, or hepatic neoplasia were consistently below 0.1 nM. The 3- or 6-hour pulse exposures to EGF (1.7 nM) between 0 to 16 hours had minimal effect on labeling index at 48 hours, but these pulse exposures at 24 or 32 hours were equivalent to continuous exposure. At 24 and 32 hours, the total specific surface binding of [125I]EGF to hepatocytes cultured free of EGF decreased to 43 and 24% of the initial values, respectively. Scatchard analysis of EGF binding indicated that hepatocytes lost all high affinity EGFRs (Kd of 0.08 nM) by 24 hours. Low affinity [125I]EGF binding at 0 hour (Kd 0.8 nM) was further reduced at 24 hours (Kd = 3.9 nM) and corresponded more closely to mitogenic concentrations of EGF in culture. These studies demonstrate that proliferative responsiveness of hepatocytes to EGF increases during culture by a process that involves prior loss of constitutive high affinity EGFRs. These results suggest that constitutive high affinity EGFRs do not elicit the proliferative response to EGF.     

Receptors for transforming growth factor-beta (TGF-beta) on rat lung fibroblasts have higher affinity for TGF-beta 1 than for TGF-beta 2.

Most cell types have receptors for transforming growth factor-beta (TGF-beta) and respond similarly to TGF-beta 1 and TGF-beta 2. We have demonstrated the presence of a single class of high-affinity receptors (approximately 10,000 sites/cell) for TGF-beta 1 (Kd = 23 pM) and TGF-beta 2 (Kd = 41 pM) on early-passage rat lung fibroblasts (RLF). Incubation with unlabeled TGF-beta 1 and TGF-beta 2 resulted in concentration-dependent inhibition of binding of 15 pM [125I]TGF-beta 1 (ED50, 20 and 28 pM, respectively) and [125I]TGF-beta 2 (ED50, 36 and 56 pM, respectively). TGF-beta receptors affinity-cross-linked with 100 pM [125I]TGF-beta 1 or [125I]TGF-beta 2 were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited labeled protein bands of 68, 88, and 286 kD. Densitometric analysis of the resulting autoradiograms showed that the different molecular weight TGF-beta binding proteins exhibited separate affinities for the two forms of TGF-beta. Both TGF-beta 1 and TGF-beta 2 altered the morphology and cytoskeleton of RLF in a similar manner, but TGF-beta 1 was more potent than TGF-beta 2 in the inhibition of RLF growth and colony formation, with 50% inhibition by 0.12 pM TGF-beta 1 and 4.4 pM TGF-beta 2. Different affinities for the TGF-beta s may indicate selectivity among the receptor subtypes with regard to the biologic responsiveness of RLF to TGF-beta s. We believe this to be the first demonstration of biologically responsive TGF-beta receptors with different affinities for TGF-beta 1 and TGF-beta 2 on cells derived from normal, nonimmortal RLF. In establishing the basic mechanisms of pulmonary fibrosis, it will be essential to understand the biology and biochemistry of the receptors that may control cell division and production of extracellular matrix components by fibroblasts.     

Alpha-macroglobulin secreted by alveolar macrophages serves as a binding protein for a macrophage-derived homologue of platelet-derived growth factor

Evidence is presented to support our hypothesis that an alpha-macroglobulin (alpha M) produced by lung macrophages serves as a specific binding protein for platelet-derived growth factor (PDGF) from these same macrophages. Culture medium "conditioned" by alveolar macrophages was fractionated by gel filtration according to molecular weight. Proteins larger than 200 kD were bound to greater than 50% of the macrophage-derived PDGF (MD-PDGF) that was extractable by 1 M acetic acid. Another approximately 25% was bound to fractions at approximately 150 kD, and approximately 20% remained unbound. The two high molecular weight fractions inhibited approximately 40% of specific [125I]PDGF binding to rat lung fibroblasts, whereas other fractions did not block PDGF binding to its receptor. Only the greater than 200 kD fractions inhibited the binding of PDGF antisera to purified human PDGF by 20% of control and exhibited specific complex formation and coelution on a gel filtration column with [125I]PDGF. The macrophage-derived alpha M (MD-alpha M) was separated from other macrophage-derived proteins by nickel-affinity chromatography and exhibited clear characteristics of alpha Ms, i.e., cross-reactivity with antibodies to human alpha 2-macroglobulin (alpha 2M) on immunoblots as well as gel migration corresponding to the electrophoretic mobility of the protease-bound "fast" and protease-unbound "slow" forms of human alpha 2M. Nickel-bound protein identified as an alpha M was bound to greater than 50% of the acid-extractable MD-PDGF in macrophage-conditioned medium, supporting the view that the greater than 200 kD protein separated by gel filtration is an alpha M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of tumor necrosis factor alpha to activated forms of human plasma alpha 2 macroglobulin.

We tested the hypothesis that human plasma alpha 2 macroglobulin (alpha 2M) is a latent binding glycoprotein for human tumor necrosis factor alpha (TNF-alpha). Human recombinant 125I-TNF-alpha was incubated for 2 hours (37 degrees C) with purified native alpha 2M and with alpha 2M that was modified by reaction with methylamine or various proteinases. 125I-TNF-alpha/alpha 2M complexes were detected by nondenaturing polyacrylamide gel electrophoresis after autoradiography or by liquid chromatography on Superose-6. 125I-TNF-alpha bound strongly but noncovalently to alpha 2M-plasmin and alpha 2M-methylamine. There was minimal binding of 125I-TNF-alpha to native alpha 2M, alpha 2M-trypsin, or alpha 2M-thrombin. A 10(6) molar excess of porcine heparin did not reduce the binding of 125I-TNF-alpha to alpha 2M-methylamine or alpha 2M-plasmin. alpha 2M-plasmin or alpha 2M-methylamine added to human plasma or serum preferentially bound 125I-TNF-alpha in the presence of native alpha 2M. 125I-TNF-alpha also bound to 'fast' alpha-macroglobulins in methylamine-reacted human, rat, mouse, swine, equine, and bovine plasma. However, TNF-alpha, preincubated with either alpha 2M-plasmin or alpha 2M-methylamine, remained a potent necrogen for cultured L929 cells. Purified 125I-TNF-alpha/alpha 2M-plasmin complex injected intravenously in CD-1 mice rapidly cleared from the circulation, unless the alpha 2M-receptor pathway was blocked by coinjection of excess alpha 2M-trypsin. These findings demonstrate that alpha 2M is a latent plasmin-activated binding glycoprotein for TNF-alpha and that TNF-alpha/alpha 2M-plasmin complexes can be removed from the circulation by the alpha 2M-receptor pathway. This suggests that alpha 2M may be an important regulator of the activity and distribution of TNF-alpha in vivo.     

Interleukin-2 directly increases albumin permeability of bovine and human vascular endothelium in vitro.

The direct effects of interleukin-2 (IL-2) on albumin permeability of cultured bovine pulmonary artery endothelial cell (BPAEC) and human arterial endothelial cell (HAEC) monolayers were studied. BPAEC were exposed to IL-2 (500 to 25,000 U/ml) for 4 h. The steady-state transfer rate of [125I]albumin across the BPAEC monolayer was 3.3 +/- 0.4%/h (n = 10) in control BPAEC (diluent alone), was significantly increased in BPAEC exposed to 500 U/ml of IL-2 (72 +/- 3% above control values, n = 6, P less than 0.02), and further increased in BPAEC exposed to 5,000 U/ml (60 +/- 2% increase above 500 U/ml values, n = 5, P less than 0.02). No further increase was noted after exposure to 25,000 U/ml of IL-2. Additionally, no further increase in [125I]albumin transfer rates was noted in BPAEC exposed to 5,000 U/ml of IL-2 for 24 versus 4 h. Similar changes were found using HAEC. Preincubation of HAEC with an anti-IL-2 low-affinity receptor antibody (anti-IL-2R alpha) inhibited the IL-2-induced permeability increase. Expression of IL-2R alpha receptors in HAEC incubated with 5,000 U/ml of IL-2 for 4 h was also found. Thus, IL-2 appears to have a direct effect on cultural arterial endothelial monolayers not requiring the presence of other cell types or serum proteins. IL-2-induced increases in endothelial macromolecular permeability may play an important role in the pathogenesis of the IL-2-induced vascular leak syndrome seen in vivo.     

Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin.

A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited.     

Distribution and Characterization of Autoantibodies to Interleukin 1α in Normal Human Sera

Antibodies against IL-1 alpha were detected in sera of apparently healthy individuals. The immunoglobulins belonged to the IgG class, particularly IgG1, IgG2, and IgG4. [125I]rIL-1 alpha bound to Fab fragments of IgG, and IgG immune complexes of molecular weights from 160 to 700 kDa were formed in the sera by [125I]rIL-1 alpha. The occurrence of detectable anti-IL-1 alpha IgG in sera of 32 male and 32 female donors was 25 and 22% respectively. As judged by Scatchard analysis of the binding data, the capacity and avidity of binding were greater in the male than in the female sera (mean capacity to bind [125I]rIL-1 alpha: 10 [0.7-27] versus 3.3 [0.5-7.3] ng/ml; and mean Kd: 5.5 [5-7] versus 11 [4-16] pM). The antibodies did not cross-bind human recombinant IL-1 beta, IL-2, IL-6, or tumour necrosis factor alpha (TNF-alpha). It is concluded that native IL-1 alpha seems to trigger production of specific, high-avidity IgG antibodies in a relatively large number of normal individuals. These autoantibodies may regulate immunoinflammatory processes involving IL-1 alpha.     

Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: role of the multifunctional alpha 2- macroglobulin receptor in antigen presentation by monocytes

Chagas' disease patients (CDP) develop both humoral and cellular immune responses against the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. Here we demonstrate that complexes formed by cruzipain and alpha 2-macroglobulin (alpha 2M) are efficiently internalized by human monocytes, and that this process results in enhanced presentation of cruzipain peptides to CD4+ T cells from CDP. Purified or serum alpha 2M binds to polymorphic cruzipains, but only a fraction of the proteinases become covalently linked. Once bound to alpha 2M, fluorescein-labeled cruzipain (FITC-cruzipain) or [125I]cruzipain were more efficiently internalized by normal peripheral blood mononuclear cells (PBMC) or monocytes; this effect was abolished by (I) pre-treating the cells with receptor-associated protein (rRAP), a known antagonist the of alpha 2M receptor (alpha 2MR/LRP), and (II) inactivating [125I]cruzipain's active site prior to the reaction with alpha 2M, indicating that the exposure of receptor binding sites on alpha 2M complexes required bait region cleavage. We then sought to determine if the alpha 2MR/LRP-dependent uptake of alpha 2M:cruzipain by monocytes resulted in increased CD4+ T cell responses of PBMC-CDP (n = 13). These effects were only revealed after depletion of CD19+ B lymphocytes from PBMC-CDP; the threshold of T cell stimulation was far lower in cultures stimulated with alpha 2M:cruzipain, as compared to antigen alone. Myocardial specimens from CDP with chronic myocardiopathy (three necropsies) were analyzed by immunohistochemistry with mAb anti-cruzipain or anti-alpha 2MR/LRP (CD81+). Extracellular depots of cruzipain were localized amidst inflammatory mononuclear infiltrates, part of which contained CD91+ macrophage-like cells. Ongoing studies should clarify if T. cruzi cysteinyl proteinases play a role in the pathogenesis of Chagas' heart disease.      

Association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens

The association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens was analyzed by a direct binding assay using radiolabeled beta 2-microglobulin and an immunoadsorbent containing a monoclonal antibody to the HLA-A,B heavy chains. Binding of beta 2-microglobulin to HLA-A,B heavy chains could be saturated with respect to the amount of membrane glucoprotein in the system and reached steady state after 6 h at 37 degrees C. Inhibition of [125I]beta 2-microglobulin binding to HLA-A,B heavy chains by beta 2-microglobulin purified from human urine or bovine colostrum resulted in identical inhibition curves and apparent dissociation constants of 1 X 10(-8) M. This evidence suggests that beta 2-microglobulins from different species have similar binding sites for HLA-A,B heavy chains.     

Uptake of 1-deoxy-1-[125I]iodo-D-mannoheptulose by different cell types: in vitro and in vivo experiments

D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty min after the intravenous injection of [125I]MH, the radioactive content of selected organs displayed the following hierarchy: muscle相似文献   

The evaluation of a radioimmunoassay for phenothiazines and thioxanthenes using an iodinated tracer

Antisera have been raised in rabbits to the immogen 2-trifluoromethylphenothiazine-10-beta-propionate bovine serum albumin. An [125I]-labelled tyrosine methyl ester derivative of the immunogen precursor has been synthesised and used with the antisera to develop a simple, precise and sensitive radioimmunoassay for phenothiazines and thioxanthenes bearing 2-trifluoromethyl substituents. The assay can detect 0.4 ng/ml of fluphenazine, trifluorperazine or (Z)-flupenthixol in 100 microliter of human serum without interference from their sulphoxide or 7-hydroxylated metabolites. An acceptable correlation between this assay and an established fluphenazine radioimmunoassay using commercial [3H]fluphenazine has been obtained for plasma samples.     

Identification of plasma membrane proteins involved in the hepatocyte invasion of Plasmodium falciparum sporozoites

To determine whether surface proteins of hepatocytes might be involved in the sporozoite invasion, plasma membrane proteins were prepared from human livers with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate) and radiolabelled with 125I (Iodogen; 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril). The labelled proteins were incubated with Plasmodium falciparum sporozoites and cross-linked with DSP (dithio-bis-succinimidylpropionate). Radiolabelled proteins released by reduction after repeated washing of the sporozoite-complex were separated by SDS-PAGE and autoradiographed. Two human hepatocyte membrane proteins of 20 and 55 kDa were found to be involved in the initial binding of P. falciparum sporozoites. The electrophoretically purified 20- and 55-kDa proteins both inhibited the binding of the corresponding radiolabelled proteins to P. falciparum sporozoites and reduced the invasion of sporozoites in an in vitro assay. We propose that these 20-kDa and 55-kDa proteins represent putative human hepatocyte receptors for P. falciparum sporozoite invasion.     

A new method for measuring antibody using radiolabeled protein A1 in a solid-phase radioimmunoassay.

A micro solid-phase radioimmunoassay was developed which utilizes radiolabeled staphylococcal Protein A ([125I]Protein A) in place of radiolabeled anti-immunoglobulin ([125I]anti-IgG) for the measurement of antibody. For the assay, antigen is adsorbed to the wells of a microtiter plate followed by dilutions of serum and [125I]-Protein A in subsequent steps. We found that this assay can be used to measure antibody (Ab) against a variety of antigens in human and rabbit but not goat immune serum. Binding of [125I]Protein A and [125I]anti-IgG to human and rabbit IgG was comparable. It was possible to quantify this amount of Ab in human serum by reference to immune rabbit serum. The sensitivity of this assay for rabbit antibody was 1 ng/ml.     

The accumulation of type I collagen mRNAs in human embryonic lung fibroblasts stimulated by transforming growth factor-beta

We examined the expression of type I collagen mRNAs (alpha 1(I) and alpha 2 (I)) by embryonic lung fibroblasts in cultures treated with transforming growth factor-beta (TGF-beta). TGF-beta caused a concentration dependent increase in the expression of alpha 1(I) mRNA for type I collagen. TGF-beta at low concentration (0.1 ng/ml) slightly increased the level of alpha 1(I) mRNA. Higher concentrations of TGF-beta (1.0 and 5.0 ng/ml) further increased the amount of alpha 1(I) mRNA. The increase in alpha 1(I) mRNA was associated with a marked increase in production of intact type I collagen molecules. TGF-beta did not increase expression of alpha 2(I) mRNA. The alpha 2(I) mRNA levels in human lung fibroblast cultures were not affected by varying the duration of exposure to TGF-beta nor the concentration of TGF-beta. In contrast, TGF-beta increased the amount of both alpha 1(I) and alpha 2(I) mRNA in NIH3T3 cells. These data suggest that the amount of alpha 2(I) mRNA is not rate limiting with respect to type I collagen production during TGF-beta stimulation in human lung fibroblast cultures.     

In vitro induction of hepatocyte synthesis of the acute phase reactant mouse serum amyloid P-component by macrophages and IL 1

The in vitro culture conditions for the induction and synthesis of the mouse acute phase reactant, serum amyloid P-component (SAP), were established using isolated hepatocytes. SAP synthesis was five to eight times greater with hepatocytes isolated from mice during the acute phase of inflammation vs. hepatocytes obtained from untreated mice. The induction of SAP synthesis in normal hepatocytes for LPS-unresponsive mice was macrophage dependent. Activated macrophages provided the most "helper" activity for SAP production. Partially purified mouse IL 1 from the P388D1 macrophage line also induced SAP synthesis. Only four IL 1 units/ml were required for optimal SAP induction. The addition of IL 1 in the presence of elicited macrophages provided an additive effect on hepatocyte SAP synthesis. The SAP-inducing activity of IL 1 copurified with its thymocyte-stimulating activity and was associated with a 11 to 25-Kd MW polypeptide. Phenylglyoxal treatment of IL 1 inactivated its thymocyte stimulating activity but not its SAP inducing potential. Inhibition of m-RNA synthesis, protein synthesis, N-glycosylation, and protein secretion effectively prevented in vitro hepatocyte SAP production.