Mouse hepatocyte synthesis and induction of the acute phase reactant: serum amyloid P-component

A methodology for obtaining reproducible in vitro induction of the synthesis of the acute phase reactant serum amyloid P-component (SAP) by purified mouse hepatocytes was established. Optimal hepatocyte culture conditions for the induction and synthesis of SAP required certain hormones, a substratum for cell attachment, and activated macrophages. Leibowitz L15 medium had to be supplemented with dexamethasone, indomethacin, insulin, glucose, and fetal bovine serum. Purified mouse IL 1 could substitute for activated macrophages in the induction of SAP. Hepatocytes were allowed to adhere to a collagen matrix to enhance both cell viability and SAP synthesis induced by IL 1. Elicited macrophages cultured with hepatocytes were capable of augmenting SAP synthesis in the presence of IL 1.     

Induction and regulation by monokines of hepatic synthesis of the mouse serum amyloid P-component (SAP)

The in vitro synthesis by mouse hepatocytes of the major acute-phase reactant, serum amyloid P-component (SAP), was induced either by inflammatory macrophages or by the addition of monokine(s), including IL 1. A single cell assay for enumerating SAP-secreting hepatocytes was developed. An increase in the frequency of SAP-synthesizing hepatocytes was found during the acute phase of inflammation. Macrophages elicited with a sterile inflammatory agent, when cultured with hepatocytes, both induced new SAP synthesis by the hepatocytes and increased the number of SAP-producing hepatocytes by sevenfold. Inflammatory macrophage culture supernatants induced new SAP synthesis in hepatocytes; however, the inducing activity did not correlate with the IL 1-dependent thymocyte-proliferating activity. Purified IL 1 alone increased SAP production without increasing the number of hepatocytes secreting SAP. A mixture of purified IL 1 with non-IL 1 monokines both increased the number of SAP synthesizing hepatocytes and the amount of SAP secreted per cell. Two non-IL 1 monokines of 70 to 80 Kd and 30 to 40 Kd were responsible for hepatocyte induction. The inducing activity was not neutralized by anti-mouse IL 1 antibody. IL 1 did contribute to the acute phase response by inducing more SAP synthesis per hepatocyte. The findings suggest that both the induction of nonsynthesizing hepatocytes into new SAP synthesis and the enhancement of the amount of SAP produced per hepatocyte are responsible for the increase in blood levels of SAP during the acute phase of inflammation.     

IL 1 requirement for B cell activation revealed by use of adult serum

Fetal calf serum is an essential component of the culture medium developed by Mishell and Dutton for the immunization of murine spleen cells in vitro. Serum from adult donors (mouse, human, rabbit) does not support antibody synthesis in this system. This "deficiency" of adult serum can be overcome with IL 1. Adult serum in the presence of IL 1 is as effective in stimulating a B cell response against xenogeneic red cells as fetal calf serum. We attribute the capacity of fetal calf serum to support an immune response in the absence of exogenous IL 1 to serum factors that cause macrophages to release IL 1 endogenously. Our findings strengthen the notion that IL 1 plays an essential role in the process of B cell activation and suggests that the use of fetal calf serum should be avoided in studies concerned with the function of interleukin 1.     

Macrophage activation-associated proteins. Characterization of stimuli and conditions needed for expression of proteins 47b, 71/73, and 120

The expression of cellular proteins was analyzed by two-dimensional gel electrophoresis during and after exposure of mouse macrophages to either mouse rIFN-gamma or natural MuIFN-beta sufficient to prime macrophages for tumor cell killing. The reversible inhibitor of protein synthesis, cycloheximide (CY), was included in some experiments during exposure to IFN. While it was present, CY suppressed protein synthesis by greater than 90%, but did not affect priming for tumor cell killing that was induced by either kind of IFN, as measured in cytotoxicity assays. Further analysis showed that, after CY and IFN were removed, protein synthesis recovered fully within 1 h. p47b, a protein that has been associated closely with the induction of the primed state in mouse macrophages, was then substantially expressed despite no new stimulation by IFN. Thus, macrophages in which protein synthesis had been reversibly inhibited delayed full processing of a signal delivered by IFN, until after protein synthesis had resumed. Such a delay in processing may explain how macrophages subsequently became activated, despite treatment with CY. The expression of the protein doublet, p71/73, was induced, regardless of which of three dissimilar agents (LPS, heat killed Listeria monocytogenes, poly I:C) was used to trigger the expression of cytolytic activity by primed macrophages. Therefore, the likelihood was increased that p71/73, expressed with p47b, is a valid phenotypic marker for fully activated, cytolytic macrophages. By contrast, p120, another protein that has been proposed as a marker of full activation in peritoneal macrophages, was expressed by bone marrow culture-derived macrophages regardless of whether or not they were cytolytic for tumor cells. It cannot be regarded as a reliable marker of macrophage activation in all circumstances, therefore.     

Induction by lipopolysaccharide of intracellular and extracellular interleukin 1 production: analysis with synthetic models

An attempt was made to identify the molecular structures that are present in bacterial LPS and induce the production of intracellular and extracellular pools of IL 1 by peritoneal macrophages of the mouse and by human monocytes. Activities of glycolipids and carbohydrates prepared by synthesis, and structurally related to the hydrophobic (Lipid A) and to the polysaccharide (PS) regions of LPS were compared with those induced by Bordetella pertussis endotoxin and by fragments derived therefrom. Both isolated regions of this LPS (PS and Lipid A) were able to induce IL 1 synthesis by monocytes and macrophages. Among the synthetic glycolipids employed, propyl-2-deoxy-2-[(3R)-3-hydroxytetrade-canamido]-4-O-pho sph ono-6-O-tetradecanoyl-beta-D-glucopyranoside (glycolipid M9) induced IL 1 secretion more efficiently than Lipid A and LPS, whereas the amounts of intracellular IL 1 produced upon induction by these three substances were comparable. Macrophages from C3H/HeJ mice were unresponsive to Lipid A and to glycolipid M9, but produced IL 1 when incubated with PS or with a hydrophilic fragment isolated after methanolysis of the endotoxin. However, all synthetic derivatives of 3-deoxy-D-manno-2-octulosonic acid (KDO) used in this study failed to induce IL 1 production by both mouse macrophages and human monocytes. The implications of these findings for a more precise comprehension of the molecular mechanism of LPS-induced activation of macrophages, and the relations between the molecular structures required for the induction of IL 1 production vs cytostatic activity in macrophages, are discussed.     

IL‐1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence‐associated secretory phenotype

Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.     

Regulation of hepatic acute phase protein synthesis by products of interleukin 2 (IL 2)-stimulated human peripheral blood mononuclear cells

Cancer patients injected with recombinant human IL 2 develop marked changes in serum concentrations of hepatic acute-phase proteins. To determine if this acute-phase response involves a change in the rate of hepatic protein synthesis and if it is due to a direct effect of IL 2 on hepatocytes, human hepatoma-derived hepatocytes (Hep-3B cells) were incubated in medium containing IL 2 or in culture supernatants from IL 2-activated human peripheral blood mononuclear cells (PBMNC). The rate of synthesis of two acute-phase proteins, complement protein factor B and albumin, was determined by the incorporation of a radiolabeled amino acid precursor into newly synthesized protein as measured by analytical gel electrophoresis of immunoprecipitates. IL 2 in concentrations from 1 to 1000 U/ml had no effect on the synthesis of factor B or albumin; conversely, there was a dose-dependent increase in the rate of synthesis of factor B and decrease in albumin synthesis mediated by culture supernatants of IL 2-activated PBMNC. The magnitude of the effect of acute-phase protein synthesis was dependent on the IL 2 concentration used for the activation of PBMNC. The rate of factor B synthesis increased approximately 4.0-fold in the presence of culture supernatants of PBMNC activated with either opsonized heat-killed Staphylococcus albus or with 1000 U/ml IL 2. Preincubation of the IL 2-activated PBMNC culture supernatants with an antiserum specific for recombinant IL 1-beta completely neutralized the capacity of the supernatants to stimulate factor B synthesis, whereas antisera specific for human IL 1-alpha or for tumor necrosis factor had no effect. These results indicate that the indirect effect of IL 2 on hepatic acute phase protein synthesis is mediated by IL 1-beta.     

Interaction of recombinant IL-1 and recombinant tumor necrosis factor in the induction of mouse acute phase proteins

Recombinant mouse and human IL-1 (alpha and beta forms), as well as rTNF-alpha when administered in vivo, induced the production of the mouse acute phase reactants: serum amyloid P-component (SAP), C3, and fibrinogen. The SAP response to all three rIL-1 proteins reached a maximum at a dose of 10(4) U/mouse, which corresponds to 1 to 10 micrograms of protein. The maximum in vivo response consisted of a 10-fold increase in SAP levels, a 2-fold increase in C3 levels, and a 3-fold increase in fibrinogen concentration. By contrast, rTNF-alpha induced a much smaller acute phase (AP) protein response (4-fold increase in SAP) when administered in vivo. Administration of a combination if rIL-1 and rTNF resulted in an AP response that was additive for SAP, synergistic for fibrinogen, but resulted in only the same amount of C3 induced by IL-1 alone. Both recombinant monokines induced new SAP synthesis by isolated hepatocytes in vitro with an optimal response occurring with either 1 U of rIL-1/ml per 2 x 10(5) hepatocytes or 10(-3) U/ml of rTNF. The hepatocyte response to IL-1 was of the same magnitude as the response of intact mice; however, the response to TNF was approximately 10(4) times more efficient in vitro. A mixture of the monokines induced an in vitro SAP response that was additive when suboptimal doses of rIL-1 were combined with optimal amounts of rTNF-alpha. Overall, the findings indicate that both monokines directly trigger hepatocyte synthesis of SAP and that their combined effect probably accounts for a substantial portion of the synthesis of these AP proteins in mice.     

Regulation of B cell tolerance by macrophage-derived mediators: antagonistic effects of prostaglandin E2 and interleukin 1

A role for prostaglandins in the mechanism of B cell tolerance induction in normal adult mouse spleen cells was examined. Two inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism, indomethacin and acetylsalicylic acid, abrogated hapten-specific B cell tolerance induction by trinitrophenyl-human gamma-globulin. Tolerance was fully restored by the addition of prostaglandin E2 (PGE2) at a concentration of greater than or equal to 6 nM. T cell-depleted spleen cells produced comparable amounts of PGE2 in culture, indicating that the tolerance promoting activity of PGE2 occurred with physiologically relevant concentrations. Depletion and reconstitution experiments indicated that macrophages in the spleen cell preparations completely accounted for both PGE2 production and the effects of indomethacin and acetylsalicylic acid on B cell tolerance induction. The macrophage product interleukin 1 (IL 1) was also found to alter B cell susceptibility to tolerance induction. Thus, human IL 1 containing monocyte supernatants and purified IL 1 were found to interfere with B cell tolerance induction when added to macrophage- and T cell-depleted splenic B cells. Tolerance was restored in such cultures by the addition of 10 nM PGE2. These experiments demonstrate that within mixed lymphoid populations macrophages through the release of mediators modulate B cell susceptibility to tolerance induction.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

The induction of lymphokine synthesis and cell growth in IL 3-dependent cell lines using antigen-antibody complexes

Several IL 3-dependent murine bone marrow-derived cell lines can be stimulated to grow with antigen-antibody (Ag.Ab) complexes. The Ag.Ab complexes induced lymphokine gene expression and the synthesis of IL 2, GM-CSF, IL 3, and BSF-1 (IL 4). The lymphokines produced by these IL 3-dependent cells appeared to stimulate their own growth, as both IL 3 and BSF-1 (IL 4) stimulated the growth of IL 3-dependent cells. Ag.Ab complexes also stimulate the growth of primary cultures of bone marrow cells that have been previously activated with IL 3. Normal bone marrow, IL 2-, and GM-CSF-dependent bone marrow cell lines could bind Ag.Ab complexes, but binding did not result in the induction of lymphokine synthesis or cell growth. Hyperimmune serum from mice also stimulated lymphokine synthesis and cell growth in IL 3-dependent cells, and the stimulatory activity was removed by treatment with Staphylococcus aureus protein A, suggesting the presence of Ag.Ab complexes.     

The expression of interleukin-6 by a rat macrophage-derived cell line

A stable rat macrophage-derived cell line (RMSV1) was established by transformation of primary peritoneal exudate cells with the SV40 virus. The RMSV1 cell line was used as a model to study the regulation of the interleukin-6 (IL6) gene expression in rate macrophages with respect to lipopolysaccharides (LPS), interleukin-1 (IL1) and glucocorticoids. The IL6 mRNA level in RMSV1 cell lines was induced 20-fold within 4 h by LPS, whereas IL1 had no effect. The glucocorticoids were able to inhibit completely the induction of the IL6 mRNA synthesis by LPS, indicating the negative regulation of the IL6 gene expression by glucocorticoids.     

Enhanced interleukin 1 (IL-1) production mediated by mouse serum amyloid P component

Purified serum amyloid P component (SAP), the major acute-phase reactant of mice, induces enhanced interleukin 1 (IL-1) production by elicited monocytes/macrophages in vitro. SAP also enhanced IL-1 elaboration by macrophages from lipopolysaccharide (LPS)-low responder mice and in the presence of polymyxin B, indicating that the small amounts of LPS present in the SAP preparation did not augment IL-1 production. Concentrations of SAP of 0.1 to 10.0 micrograms/ml enhanced IL-1 production by elicited and bacillus Calmette-Guerin (BCG)-activated peritoneal macrophages, but not by resident peritoneal macrophages. The inflammation-induced monocyte/macrophage population displayed selective binding of SAP. The mouse macrophage line P388D1, also could bind SAP and display enhanced IL-1 production in response to SAP. SAP did not bind to the macrophage cell line RAW264.7 nor did it enhance IL-1 secretion by this line. The results suggest that this acute-phase reactant has the potential to enhance inflammatory and immunological events mediated by IL-1.     

Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.     

Humoral primary immune response in vitro in a homologous mouse system: replacement of fetal calf serum by a 2-mercaptoethanol or macrophage-activated fraction of mouse serum.

The primary immune response in mouse spleen cell cultures against heterologous red cell antigens is dependent on the medium being supplemented with selected batches of fetal calf serum. Mouse serum itself is not able to support this response. The active immune response-supporting component in fetal calf serum seems to be a distinct factor (s), which has been partially purified by Sephadex G-100 filtration and termed MaSF-2-mercaptoethanol-activated serum factor. In this report it is demonstrated that MaSF is also present in mouse serum. For functional detection, mouse MaSF has to be separated from higher m.w. inhibitors, and has to be activated by 2-ME. After separation and activation mouse MaSF can support the primary immune response in a completely homologous in vitro culture system. Evidence is presented that MaSF can also be activated by macrophages. It is concluded that macrophages and 2-ME have the same mode of action in the primary immune response in vitro, i.e., induction of lymphocyte competence by activation of a serum factor.     

Stimulation of liver functions in hierarchical co-culture of bone marrow cells and hepatocytes

A hierarchial co-culture, in which rat hepatocytes and non-parenchymal liver cells (NPLCs) were separated by a collagen layer and which was designed to mimic the in vivo microenvironment, was carried out with the aim of developing a module for bio-artificial liver support. Compared with a monolayer co-culture and hepatocytes cultured alone in a monolayer, higher urea synthesis activity was maintained for 6 d in the hierarchical co-culture. When a rat hepatoma cell line H4-II-E-C3, which retains the induction of tyrosine aminotransferase (TAT), was co-cultured in a monolayer with NPLCs, dose-dependent stimulation of TAT induction was observed. In a hierarchical co-culture, NPLCs further stimulated TAT induction in H4-II-E-C3 cells. Since peritoneal macrophages could stimulate TAT induction in hepatocytes in both monolayer and hierarchical co-cultures, bone marrow cells, which can proliferate and differentiate into macrophages in vitro, were investigated as a possible substitute for NPLCs. Bone marrow cells isolated from rat femurs were cultivated in the presence of IL-3 and macrophage colony-stimulating factor (M-CSF), and co-cultured with hepatocytes. Urea synthesis and TAT induction of hepatocytes were stimulated in the co-culture. The co-culture of bone marrow and H4-II-E-C3 cells, both of which have proliferation ability in vitro, was also shown to be effective in stimulating liver functions. The hierarchical configuration, in which two cell types can communicate with the soluble factor(s) through a collagen layer, was found to be more effective than a monolayer in long-term co-culture.     

Adiponectin differentially regulates cytokines in porcine macrophages

Adiponectin, an adipocyte-derived hormone, attenuates the production of TNFalpha by activated human macrophages. In the present study, we used porcine blood-derived macrophages to test the hypothesis that the anti-inflammatory action of adiponectin includes suppression of IL6 and an induction of IL10. Adiponectin suppressed both TNFalpha and IL6 production in macrophages activated with lipopolysaccharide (P<0.01). In contrast, adiponectin increased IL10 expression (P<0.05) and augmented (P<0.05) the induction of this cytokine by lipopolysaccharide (LPS). Mechanistically, the attenuation of proinflammatory cytokine production by adiponectin was associated with an attenuation of the translocation of NFkappaB to the nucleus. Either adiponectin or inhibition of ERK1/2 with U0126 diminished the induction of IL6 by LPS (P<0.05), but the combination of adiponectin and the inhibitor did not further reduce IL6 production. In contrast, the inhibitory actions of adiponectin and a p38 MAPK inhibitor (SB203580) were additive (P<0.05). These data indicate that the anti-inflammatory actions of adiponectin include suppression of IL6 and induction of IL10. In addition, we provide evidence that some of the anti-inflammatory actions of adiponectin are mediated in part by suppression of NFkappaB signaling and ERK1/2 activity.     

Down-regulation of interleukin 1 production by macrophages of sarcoma-bearing mice

Peritoneal macrophages from mice bearing a transplantable methylcholanthrene-induced sarcoma produced progressively less IL 1 as tumor burden increased. The loss of activity was not explained by the production of any inhibitor of the mouse thymocyte comitogen bioassay. Immune precipitation with a polyclonal antibody confirmed the decline in IL 1 appearance. Although tumor-bearing animals lost approximately 17% of their carcass mass, the reduced production of IL 1 was not satisfactorily explained by coexistent malnutrition, since similarly depleted non-tumor-bearing mice were capable of producing IL 1. In addition to an altered IL 1 production by macrophages of tumor-bearing mice, SDS-polyacrylamide gel electrophoresis and autoradiography revealed that the pattern of secretory protein synthesis from LPS-stimulated and unstimulated peritoneal macrophages differed between tumor-bearing and control animals. Administration of LPS to tumor-bearing mice early after tumor transplantation resulted in reduced tumor growth and prevented the down-regulation of in vitro IL 1 production by peritoneal macrophages. These findings demonstrate a specific defect in IL 1 production associated with increasing tumor burden. Further studies are required to determine whether this defect in IL 1 synthesis contributes to the increased tumor growth.     

Stimulation of rat mesangial cell proliferation by macrophage interleukin 1

Conditioned media from LPS-activated rat peritoneal macrophages enhanced the proliferation rates of cultured rat glomerular mesangial cells. This macrophage-derived activity extensively co-purified with interleukin 1 (IL 1) activity through sequential ammonium sulfate precipitation, S-200 gel chromatography, DEAE-cellulose anion exchange chromatography, and phenyl-Sepharose chromatography. In addition, the macrophage-derived factor was heat-labile (80 degrees C) and inactivated by phenylglyoxal, thus allowing tentative identification as IL 1. Macrophage supernatants and purified IL 1 enhanced the proliferative rates of mesangial cells only in the presence of serum; the use of platelet-poor plasma or serum depleted of platelet-derived growth factor was without effect. IL 1 acted to increase the percentage of cycling cells, without a change in the length of the individual cell cycle times. These findings provide a potential mechanism whereby activated macrophages, in combination with platelet factors, enhance mesangial cell proliferation. Such processes may contribute to the mesangial hypercellularity frequently found in immune-mediated glomerulonephritis.     

Sodium periodate-induced T cell mitogenesis: an analysis of the requirement for Ia and IL 1

T lymphocytes oxidized with the mitogen sodium periodate undergo a proliferative response when cultured in the presence of Ia+ accessory cells. However, the exact role(s) the accessory cells play in such a response has not been clearly defined. We have evaluated the role of Ia and the requirement for interleukin 1 (IL 1) in periodate mitogenicity by using the Ia+ cloned tumor cell lines P388AD (Ia+, IL 1 inducible) and P388NA (Ia+, IL 1 noninducible) as accessory cells. P388AD but not P388NA was able to supply accessory cell function to periodate-treated T cells, suggesting that Ia expression alone was not sufficient to reconstitute a response. Monoclonal anti-I-Ad and anti-I-Ed antibody blocked the accessory cell function of P388AD. In addition, monoclonal antibody GK 1.5, directed against the T cell determinant L3T4a, blocked the P388AD/periodate-treated T cell interaction, confirming that this interaction was restricted by class II molecules. Although Ia expression was required, the response was not major histocompatibility complex (MHC) restricted, because allogeneic as well as syngeneic macrophages were capable of supplying accessory cell function to periodate-treated T cells. Exogenous IL 1 alone was able to trigger periodate-treated T cells, suggesting that Ia was required for the induction of IL 1 synthesis by the accessory cells. Furthermore, purified IL 2, devoid of IL 1 activity, was able to fully reconstitute the proliferative response of accessory cell-depleted oxidized T cells to a level equal to that of whole spleen accessory cells or P388AD. These data suggest that periodate-treated T cells can proliferate with IL 1 alone and that Ia+ accessory cells in periodate-mediated T cell mitogenicity may function in the release of IL 1 and the induction of IL 2 synthesis by the T cells.