Murine gamma interferon activates the release of a macrophage-derived Ia-inducing factor that transfers Ia inductive capacity

In this report we demonstrate that when the murine macrophage tumor cell line P- 388D1 is incubated for 48-72 h with either concanavalin A-stimulated rat spleen cell supernatant or cloned murine immune interferon (IFN-gamma), the cultured cells release a cell-free factor activity that in turn induces the cell surface expression of Ia antigen on the murine monocyte cell line WEHI-3. This IFN-gamma-stimulated, Ia-inducing activity cannot be blocked with an anti-IFN-gamma heteroantiserum that does block the induction of Ia expression on WEHI-3 by both cloned murine IFN-gamma and rat Con A supernatant. The Ia-inducing factor ( IaIF ) generated from P- 388D1 after stimulation by IFN-gamma does not demonstrate any antiviral activity. The P- 388D1 -derived IaIF is not shed plasma membrane Ia glycoprotein molecules, as demonstrated by the inability of the active component to bind specifically to an anti-I-Ad affinity column or to a protein A column after the active supernatant is first treated with an excess of anti-I-E/Cd,k monoclonal antibody.     

Helper signals in the plaque-forming cell response to protein-bound haptens

We have demonstrated the ability of a series of murine T cell hybridomas to deliver an antigen-specific, B cell I-region-restricted helper signal in the generation of specific PFC responses to protein-bound haptens. With some hybridomas the elicitation of optimal PFC responses required the addition of nonspecific factors provided by culture supernatants of concanavalin A-stimulated (Con A SN) spleen cells. Using hapten-primed B cells depleted of both T cells and macrophages (Mphi) we have now demonstrated a requirement for three nonspecific factor preparations to substitute for spleen Con A SN in the elicitation of optimal PFC responses. The first preparation was the interleukin 1 containing culture supernatant of the Mphi tumor cell line P388D1, the second the interleukin 2 (IL-2) and B cell growth factor containing Con A SN of the T cell hybridoma FS6-14.13, and the third, the gamma interferon containing Con A SN of the T cell hybridoma FS7-20.6.18. The P388D1 and FS6-14.13 factor preparations were most effective when added at the initiation of culture, while the FS7-20.6.18 factor preparation was most effective when added at 24 h of culture. The activity of FS6-14.13 Con A SN was depleted by incubation with the IL-2-dependent T cell line HT-2. The activity of FS7-20.6.18 Con A SN was abrogated by incubation at pH 2. The results suggest that the generation of PFC responses to protein-bound haptens require at least three nonspecific factors in addition to an antigen/Ia specific helper signal.     

Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.     

Regulation of murine macrophage Ia-antigen expression by products of activated spleen cells

This investigation examined the effects of mediators derived form activated spleen cells on macrophage Ia-antigen expression and function. Incubation of adherent thioglycollate-induced murine peritoneal macrophages(> 90% Ia-) with concanavalin A (Con A)- stimulated spleen cell supernate (Con A sup) resulted in a dose- dependent increase in the percentage of Ia-containing (Ia+) phagocytic cells, as detected by antiserum-and-complement-mediated cytotoxicity. The Ia-antigen expression of macrophages incubated with unstimulated spleen cell supernate supplemented with Con A (Control sup) declined. Pretreatment of the macrophages with anti-Ia and complement before addition of the Con A sup did not inhibit subsequent Ia-antigen expression, suggesting that Ia- macropohages were converted to Ia+ cells. These findings were not a result of adsorption of soluble Ia- antigen from the Con A sup, because Ia-antigen expression was detected by an antiserum specific for the haplotype of the macrophages but not that of the allogeneic spleen cells from which the supernate was prepared. Con A sup-cultured macrophages also stimulated the proliferation of allogeneic spleen cells significantly better than Control sup-cultured macrophages in the mixed leukocyte reaction (MLR). Pretreatment of Con A sup-cultured macrophages with anti-Ia and complement before addition of splenic responder cells abrogated their stimulatory capacity, indicating the Ia dependence of the MLR. We hypothesize that regulatory lymphokine(s) can induce both the expression of the Ia+ phenotype by macrophages and the functional capability to stimulate the MLR, and that macrophages lose these capabilities in the absence of such mediator(s).     

Contrasting effect of alpha/beta- and gamma-interferons on expression of macrophage Ia antigens

IFN-gamma is known to induce the expression of Ia antigens on macrophages. We found that murine IFN-alpha and -beta blocked the effects of IFN-gamma in a dose-dependent manner. The antagonistic effect of IFN-alpha and -beta was observed even when macrophages were prestimulated with IFN-gamma. These inhibitory effects of IFN-alpha or -beta were blocked by their respective antibodies. The block exerted by IFN-alpha/beta was similar whether Ia levels were monitored by immunofluorescence with anti-Ia mAb, or by stimulation of freshly sensitized, alloreactive T lymphoblasts. Adherent macrophage-rich populations from newborn mice were incapable of expressing Ia antigens following stimulation with IFN-gamma, and would inhibit the response of adult macrophages to this lymphokine. Addition of anti-IFN-beta mAb, but not anti-IFN-alpha allowed newborns' macrophages to express Ia in response to IFN-gamma, and ablated the suppressive activity toward adult cells. These results indicate that IFN-alpha and -beta, which can be produced in the course of self-defense responses and during ontogeny, may contribute to the down-regulation of macrophage Ia expression.     

Persistent expression of Ia antigen and viral genome in visna-maedi virus-induced inflammatory cells. Possible role of lentivirus-induced interferon

In this study we investigated the pathogenesis of the lymphoproliferative response in the chronic-active visna maedi virus-induced inflammatory lesions. Viral RNA expression was confined to macrophages, but only in tissues showing inflammatory lesions. A persistent and high level of Ia antigen expression was seen in macrophage-like cells in the inflammatory lesions, and the amounts of viral RNA and Ia expression were closely correlated. A small subpopulation of macrophages contained both viral RNA and Ia antigen, and these were found in greatest number in the lung. In vitro experiments showed that a lentivirus-induced interferon (LV-IFN) could induce Ia antigens in normal sheep spleen and lymph node cells as well as in a transformed sheep macrophage cell line. Ia antigen expression in macrophages was transient in the absence of a continuing IFN stimulus and persisted for at least 2 wk in the presence of LV-IFN. LV-IFN also restricted viral replication in macrophages. It is suggested that LV-IFN induced by the inflammatory cells in visna-maedi lesions may induce Ia antigen expression in macrophages, thereby indirectly causing the lymphoproliferative response and restricted virus replication.     

Interleukin-induced increase in Ia expression by normal mouse B cells

The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen- presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology.     

Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.

Expression of the macrophage mannose receptor is inhibited by interferon gamma (IFN-gamma), a T helper type 1 (Th-1)-derived lymphokine. Interleukin 4 (IL-4), a Th-2 lymphocyte product, upregulates major histocompatibility class II antigen expression but inhibits inflammatory cytokine production by macrophages. We have studied the effect of IL-4 on expression of the macrophage mannose receptor (MMR) by elicited peritoneal macrophages. We found that recombinant murine IL-4 enhances MMR surface expression (10-fold) and activity (15-fold), as measured by the respective binding and degradation of 125I-mannose-bovine serum albumin. Polymerase chain reaction analysis of cDNAs from purified primary macrophage populations revealed that MMR, but not lysozyme or tumor necrosis factor alpha, mRNA levels were markedly increased by IL-4. The above effects were associated with morphologic changes. These data establish IL-4 as a potent and selective enhancer of murine MMR activity in vitro. IL-4 induces inflammatory macrophages to adopt an alternative activation phenotype, distinct from that induced by IFN-gamma, characterized by a high capacity for endocytic clearance of mannosylated ligands, enhanced (albeit restricted) MHC class II antigen expression, and reduced proinflammatory cytokine secretion.     

Surface antigens of melanoma and melanocytes. Specificity of induction of Ia antigens by human gamma-interferon

IFN-gamma is known to induce expression of Ia antigens on a variety of cell types. In the present study, this activity of IFN-gamma has been analyzed with a panel of 36 melanoma cell lines, normal melanocytes, and 97 cell lines representing a range of other differentiation lineages. 55% of the melanoma cell lines express Ia antigens in a constitutive manner without IFN-gamma induction. Of the 16 Ia-melanoma lines, 13 could be induced to express Ia antigens by IFN-gamma, whereas three were noninducible. Melanocytes, which do not normally express Ia antigens, are converted to Ia expression by IFN-gamma. Ia antigens expressed constitutively or after IFN-gamma induction were identified with antibodies detecting monomorphic and allomorphic products of DR and DC loci. IFN-gamma appeared to be unique in its ability to induce Ia expression on melanoma and melanocytes; 14 other agents (including IFN-alpha and IFN-beta) known to influence growth or differentiation did not have Ia-inducing activity. Equally striking is the restriction of antigenic changes following IFN-gamma induction to HLA-associated products; of the 38 systems of cell surface antigens examined, only HLA- A,B,C, beta 2m, and Ia antigens were affected. A variety of other Ia- cell types were shown to be Ia-inducible by IFN-gamma; these included established lines of breast, colon, pancreas, bladder, kidney, ovary, and brain cancers, and cultures of normal fibroblasts, kidney epithelia, and epidermal keratinocytes. In contrast, three tumor types, teratocarcinoma, choriocarcinoma, and neuroblastoma, were not inducible for Ia expression, even though IFN-gamma could induce expression of HLA- A,B,C products. The broad representation of Ia antigens on most somatic cell types expressed either constitutively or after IFN-gamma can be viewed in an immunological context (antigen presentation/immune regulatory signals) or could indicate that Ia products have functions other than those related to immune reactions.     

Cellular immune response from Chagasic patients to CRA or FRA recombinant antigens of Trypanosoma cruzi

We propose to analyze the relation between the cellular immune response of Chagas' disease patients after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant antigens cytoplasmatic repetitive antigen (CRA) or flagellar repetitive antigen (FRA) of T. cruzi and the chronic clinical forms of disease. Cells were stimulated using phytohemagglutinin, CRA, FRA, or a soluble antigen of Epimastigota (Ag-Epi) for 24 hr, 72 hr, or 6 days. The proliferation of cells was evaluated after 6 days of culture by quantification of incorporated 3H-thymidine. Cytokines were measured in the supernatants obtained after 24 hr (tumor necrosis factor [TNF]-alpha and interleukin [IL]-4), 72 hr (IL-10), and 6 days (interferon [IFN]-gamma) using enzyme-linked immunosorbent assay (ELISA). Cells of the Chagas patients stimulated with the recombinant antigens exhibited higher proliferation responses compared with that of non-Chagas (NC) individuals. However, when proliferation was compared between patients with the cardiac form (CF) or indeterminate form (IF), it was not possible to establish a difference in the response. So far as the cytokines secreted in the culture supernatants after stimulation in vitro with T. cruzi antigens were concerned, the results showed that CRA, as well as Epi-Ag, were able to stimulate the production of TNF-alpha and IFN-gamma in Chagas patients as compared with NC individuals. However, the cytokine levels after stimulation with the T. cruzi antigens were not different between the patients with CF and IF. CRA was capable of inducing a T helper type 1 (Th1) immune response, with elevated production of TNF-alpha and IFN-gamma in Chagas patients that are carriers of CF and IF clinical forms.     

Interferon beta, a cofactor in the interferon gamma production induced by gram-negative bacteria in mice

The interferon (IFN) gamma production of splenocytes from closely related C57BL/10ScSn (Sn) and C57BL/10ScCr (Cr) mice was compared. Concanavalin A and CD3 monoclonal antibodies induced high levels of IFN- gamma in both Sn and Cr splenocytes. By contrast, treatment with gram- negative bacteria induced IFN-gamma only in Sn splenocytes; in Cr splenocytes, the IFN-gamma response was heavily impaired. The IFN-gamma induction by bacteria requires the cooperation of IFN-gamma-producing cells with macrophages. Depletion of macrophages from Sn splenocytes resulted in the loss of ability to produce IFN-gamma after bacterial stimulation. Reconstitution with new Sn macrophages restored the IFN- gamma responsiveness, whereas reconstitution with Cr macrophages failed to do so. Normal function of IFN-gamma-producing cells and a defective function of macrophages of Cr mice was demonstrated by evidence showing that whole or macrophage-depleted Cr splenocytes, when supplemented with Sn macrophages, acquire the ability to produce IFN-gamma in response to bacteria. A similar effect was achieved by supplementing Cr splenocytes with supernatants of bacteria-stimulated Sn macrophages or with recombinant murine IFN-beta or IFN-alpha. Preincubation of active macrophage supernatants with antibodies to IFN-beta suppressed the helper activity for Cr splenocytes. Moreover, the bacteria-induced production of IFN-gamma by Sn splenocytes could be inhibited by antibodies to murine IFN-beta. The results provide evidence that IFN- beta is an important cofactor of IFN-gamma induction, which is not induced in Cr mice by gram-negative bacteria.     

The delayed ontogenesis of Ia-positive macrophages: implications for host defense and self-tolerance in the neonate

We have recently compared a number of macrophage functions in new-born and adult mice. In the adult, the most efficient resistance against Listeria infection requires a number of macrophage-T lymphocyte interactions which ultimately result in activated macrophages which eliminate the bacteria. A subpopulation of adult macrophages are distinguished by the presence of cell-surface; immune-response-gene-associated (Ia) antigens. These macrophages ingest and then "present" antigen to and thus activate T lymphocytes. Extensive in vitro experiments have shown that T lymphocyte activation requires both antigen and Ia-bearing macrophages. Ia-negative macrophages are not involved in this process. The T lymphocyte thus stimulated secretes lymphokines which then activate macrophages to acquire bactericidal and tumoricidal activity. In contrast to the adult mouse, essentially no Ia-bearing antigen presenting macrophages can be found in the neonatal spleen and peritoneal cavity. Neonatal and adult macrophages were similar in all other examined aspects--phagocytic activity, cytocidal function after lymphokine stimulation, and interleukin-1 secretion after exposure to endotoxin. The absence of Ia-bearing macrophages in neonates would be expected to contribute to their increased susceptibility to Listeria. The absence of Ia-bearing macrophages in the neonate is not the result of any intrinsic defects in these cells, as they can be made to express Ia in vitro. Nor is the deficit in Ia-bearing macrophages due to the hyporeactivity of neonatal helper T lymphocytes as adult athymic mice have significant numbers of Ia-bearing macrophages. Neonatal splenocytes or their conditioned media inhibit macrophage Ia expression when transferred into adult mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

The processing and presentation of Listeria monocytogenes antigens by macrophages

Macrophages play a requisite role in the induction and expression of T lymphocyte responses to Listeria monocytogenes. For effective T cell-macrophage interaction to occur, macrophages must perform at least two fundamental functions. They must take up and handle the antigen, and they must express appropriate membrane glycoproteins encoded for by the I-region of murine major histocompatibility gene complex (Ia molecules). Data collected in a murine model suggests that the following sequential events are involved in the Listeria-macrophage-T cell interaction. Listeria interaction with macrophage cell surface via trypsin sensitive structures. Interiorization within phagosomes. Phagosome-lysosome fusion. Partial degradation of Listeria. Transfer of protein antigen fragments to macrophage cell surface. Recognition of macrophage surface antigen and I-region associated (Ia) molecules by the T cell receptor. The essential feature of this model is that: antigen handling occurs intracellularly and independently of macrophage cell surface Ia molecules. With regard to the survival advantage of this mechanism, one may speculate that the degradation of pathogens by macrophages may serve to increase the number of different structural moieties which can act as antigens. Thus, bacterial components normally sequestered in the interior of organisms could conceivably serve as antigens, and the multiplicity of such antigenic determinants would make it less likely that a nonresponder status with respect to I-region gene function would be generated. This mechanism may be especially relevant to host defense against intracellular pathogens such as Listeria monocytogenes.     

脂多糖对腹膜和骨髓巨噬细胞产生红系造血调控因子的影响

腹膜和骨髓巨噬细胞培养中含红系造血祖细胞增殖的刺激因子,它不但能提高红系祖细胞的产率,而且还可增加每个集落中细胞数。大肠杆菌脂多糖(lipopolysaccharide,LPS)可提高巨噬细胞产生红系增殖刺激因子的能力。经过LPS激活的巨噬细胞培养液对晚期红系造血祖细胞增殖表现出双向调控作用,低浓度促进,高浓度抑制,其中LPS激活腹膜贴壁巨噬细胞的活性高于骨髓巨噬细胞。     

Biological and antigenic similarities of murine interferon-gamma and macrophage-activating factor

Murine peritoneal exudate cells (PEC) treated with murine recombinant interferon-gamma (IFN-gamma) (greater than 99% estimated purity), or concanavalin A-stimulated spleen cell supernatants developed tumoricidal properties (macrophage activation factor [MAF] activity). MAF activity was found to occur with treatments of 10 U/ml IFN-gamma, and at levels as low as 1 U/ml IFN-gamma if a second signal (5 ng/ml endotoxin) was present in the MAF assay. Endotoxin (lipopolysaccharide [LPS]) alone at these levels failed to induce MAF; induction of MAF was observed at 1,000-fold greater levels. The ability of IFN-gamma to stimulate murine PEC was species specific. Various sources of materials that displayed MAF activity, including supernatants from interleukin 2- dependent cloned cytotoxic murine T lymphocyte lines that did not display detectable antiviral activity, were neutralized by antibody raised and affinity purified against recombinant IFN-gamma. Thus, IFN- gamma, although never detectable by antiviral assays, appears to be present in many lymphokine preparations and has potent macrophage activation capability.     

Obligatory role of gamma interferon in T cell-replacing factor- dependent, antigen-specific murine B cell responses

The role of gamma interferon (IFN-gamma) in T cell-replacing factor (TRF) activity for antigen-specific plaque-forming cell (PFC) responses in vitro was studied using antibodies to murine IFN-gamma (Mu IFN-gamma). TRF activity was present in supernatants (Sn) of Con A- or mixed leukocyte reaction-stimulated murine spleen cells as well as in an IL-2-rich fraction of phytohemagglutinin-stimulated human peripheral blood lymphocyte Sn and in the Sn of the Gibbon T lymphoma MLA-144. The human TRF was highly active with cells from nu/nu mice and normal mice but not with cells from animals with the xid immunologic defect, similar to the activity of murine TRF. Antibodies to IFN-gamma consisted of hyper-immune rabbit antisera, IFN-gamma affinity-purified rabbit immunoglobulin and an interspecies hybridoma specific for Mu IFN-gamma. The results show that the activities of all preparations of TRF are markedly diminished or abrogated by antibody to Mu IFN-gamma but not by antibodies to human IFN-gamma (Hu IFN-gamma), nor by normal rabbit sera or purified rabbit Ig. The degree of inhibition was dose dependent and was quantitatively reversed by the addition to the cultures of recombinant-derived Mu IFN-gamma (Mu rIFN-gamma) but not Hu rIFN-gamma. This reversal was fully antigen specific and thus not attributable to polyclonal B cell activation by IFN-gamma, which is inactive alone in the TRF assay. Kinetic analysis shows that IFN-gamma must act by 24-48 h to produce PFC responses at 4 d. Together, the data demonstrate that IFN-gamma is a necessary mediator for TRF effects and that IFN-gamma is induced by TRF from T-depleted murine spleen cells in sufficient quantity to support large antibody responses. The source of this IFN-gamma may be the potent natural killer cells that are induced in cultures stimulated with TRF.     

B cell helper factors. I. Requirement for both interleukin 2 and another 40,000 mol wt factor

A helper factor(s) distinct from interleukin 2 (IL-2) was shown to be present in the concanavalin A-stimulated supernatant of normal mouse spleen cells (normal Con A Sn). Spleen cells thoroughly depleted of T cells required both IL-2 and this factor to produce antibody-secreting cells in response to sheep erythrocytes, although in the presence of IL- 2 and a few T cells the requirement for the factor was less apparent. The factor had an apparent approximately 40,000 mol wt. The factor was found in normal Con A Sn that had been depleted of IL-2 by absorption with IL-2-dependent T cells and was absent from Con A-stimulated supernatants of the IL-2-producing T cell hybridoma, FS6-14.13. These results indicate that multiple helper factors control the B cell response to antigen and that IL-2, in addition to its T cell growth promoting activity, plays a direct role in B cell responses.     

Nature of the antigenic complex recognized by T lymphocytes II. T-cell activation by direct modification of macrophage histocompatibility antigens

In order to analyze the molecular structures involved in T-cell recognition we developed an in vitro primary response against alloantisera bound to histocompatibility antigens in which nonimmune guinea pig T cells can be sensitized and subsequently challenged in tissue culture with antisera-treated macrophages. If macrophages were incubated with alloantisera directed against the I-region-associated (Ia) antigens of the guinea pig major histocompatibility complex (MHC) T cells could be sensitized to the antisera bound to macrophage Ia determinants. Anti-Ia-treated syngeneic macrophages in the first and second cultures elicited specific T-cell activation, as measured by increased DNA synthesis, to the antisera-induced immunogenic determinants. Similarly, antiIa-treated allogeneic macrophages also specifically stimulated T cells to antisera bound to allogeneic Ia determinants while reducing the mixed leukocyte reaction. Antisera to the B.1 antigens of the guinea pig MHC, the homologue of the mouse H-2K or H-2D antigens, also elicited specific T-cell activation that did not cross-react with that produced by the anti-Ia alloantisera. Furthermore, the anti-B.1-induced stimulation appeared to be associated with the Ia antigens of the macrophage used for priming since (2 x 13)F1 T cells sensitized with anti-B.1-treated parental macrophages could be restimulated only with the parental macrophage used for initial sensitization, and not with those of the other parent. Since the parental strain 2 and strain 13 guinea pigs express serologically identical B.1 antigens and differ only by Ia antigens of the MHC, this observation suggests that both B.1 and Ia antigens may be included in the immunogenic complex recognized by T cells. However, we cannot rule out the possibility that this restriction is due to other genetic differences between strain 2 and strain 13 guinea pigs that is unrelated to the I-region. We interpret these findings as showing that macrophage Ia antigens may serve to directly present antigens bound to the Ia molecule, and possibly indirectly aid in the presentation of antigens bound to other membrane components, such as the B.1 antigens.     

Influences of gamma interferon on synovial fibroblast-like cells. Ia induction and inhibition of collagen synthesis

The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.