T-cell-mediated activation of macrophages.

Functionally diverse subpopulations of macrophages and lymphocytes, a wide array of stimulatory signals, and an enormous effector repertoire of activated macrophages keeps this field dynamically active. We review new advances in the identification of cytokines that interact to activate macrophages, and in the discovery of effector molecules used by activated macrophages to destroy their targets.

A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer.

Endogenous murine angiostatin, identified as an internal fragment of plasminogen, blocks neovascularization and growth of experimental primary and metastatic tumors in vivo. A recombinant protein comprising kringles 1-4 of human plasminogen (amino acids 93-470) expressed in Pichia pastoris had physical properties (molecular size, binding to lysine, reactivity with antibody to kringles 1-3) that mimicked native angiostatin. This recombinant Angiostatin protein inhibited the proliferation of bovine capillary endothelial cells in vitro. Systemic administration of recombinant Angiostatin protein at doses of 1.5 mg/kg suppressed the growth of Lewis lung carcinoma-low metastatic phenotype metastases in C57BL/6 mice by greater than 90%; administration of the recombinant protein at doses of 100 mg/kg also suppressed the growth of primary Lewis lung carcinoma-low metastatic phenotype tumors. These findings demonstrate unambiguously that the antiangiogenic and antitumor activity of endogenous angiostatin resides within kringles 1-4 of plasminogen.

Macrophages in resistance to rickettsial infections: protection against lethal Rickettsia tsutsugamushi infections by treatment of mice with macrophage-activating agents.

Peritoneal macrophages of BALB/c and C3H/HeN mice activated in vivo by intraperitoneal inoculation of viable Mycobacterium bovis strain BCG or the nonliving macrophage-activating agent Propionibacterium acnes (Corynebacterium parvum), were resistant to infection with Rickettsia tsutsugamushi, and they killed bacteria that did gain entry into the intracellular environment of these cells. This macrophage resistance to infection and intracellular destruction of rickettsiae was dependent upon development of an immune response to the activating agents, since macrophages elicited by sterile inflammatory agents failed to display either microbicidal activity unless cells were exposed to factors present in lymphokine-rich culture fluids from antigen or mitogen stimulated spleen cells (LK) in vitro. C3H/HeN mice that had been treated with activating agents, but not sterile inflammatory irritants, also survived intraperitoneal inoculation of up to 10(4) R. tsutsugamushi. This nonspecific protection required the chronic presence of activated macrophages: acute immune response induced by intraperitoneal injection of PPD into mice inoculated intradermally with BCG, or intraperitoneal inoculation of conconavalin A, were not sufficient to induce survival of rickettsial disease, although macrophages from these animals were activated to kill rickettsiae at the time of challenge. The critical nature of activated macrophages in nonspecific protection against rickettsial infection was demonstrated with the macrophage-defective C3H/HeJ mice. These mice are equally as susceptible as C3H/HeN mice to intraperitoneal inoculation of R. tsutsugamushi, but do not develop activated macrophages in response to BCG infection, and are not protected against lethal rickettsial challenge following BCG treatment.

Interleukin-2 suppresses activated macrophage intracellular killing activity by inducing macrophages to secrete TGF-beta.

Phorbol myristate acetate (PMA) treatment of an EL-4 thymoma cell line (EL-4FARRAR) induced secretion of a factor that inhibited intracellular killing of Leishmania major amastigotes by activated macrophages. Analysis of the cytokines produced by EL-4 cells after PMA stimulation identified interleukin-2 (IL-2, 2500 U/ml), IL-4 (1280 U/ml), interferon-gamma (IFN-gamma; 100 U/ml), and granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor beta (TGF-beta) was detected. Each of the cytokines present in EL-4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN-gamma and GM-CSF both activated macrophages to kill Leishmania; IL-2 and IL-4 had no activity for induction of this antimicrobial effector function. IL-2 and IL-4 were tested for their capacity to inhibit lymphokine- or IFN-gamma-induced destruction of L. major by macrophages: IL-4 was ineffective, but IL-2 markedly suppressed the activation of macrophages for intracellular killing. Addition of > or = 10 U/ml of IL-2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL-4 fluids with anti-IL-2 antibody resulted in > 80% reduction in suppression of intracellular killing. The suppressive effects of IL-2 were not direct, but mediated by TGF-beta. IL-2 induced resident peritoneal macrophages to secrete > 5000 pg/ml TGF-beta 1, a quantity that is > 500-fold higher than constitutive background levels (20-40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF-beta was not dependent increases in TGF-beta 1 mRNA. Treatment of cultures with EL-4 fluids or recombinant IL-2 in the presence of antibody to TGF-beta 1 blocked the suppressive activity of both. Thus, IL-2 was the major suppressor factor in EL-4 fluids, and it acted indirectly through the induction and autocrine action of TGF-beta.

Immunotherapy of tularemia: characterization of a monoclonal antibody reactive with Francisella tularensis.

An IgM monoclonal antibody (mAb) recognized surface antigens specific to Francisella tularensis wild-type (Schu4) and live vaccine strain (LVS), and reacted with both in ELISA and slide agglutination tests. This mAb also reacted with LVS microorganisms in tissues of infected mice as assessed by an indirect fluorescence technique. Western blot analysis showed the mAb to react with antigens associated with F. tularensis LPS.

Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes.

Nitric oxide (NO) produced by cytokine-treated macrophages and hepatocytes plays a vital role in protective host responses to infectious pathogens. NO inhibits iron-sulfur-dependent enzymes involved in cellular respiration, energy production, and reproduction. Synthesis of L-arginine-derived nitrite (NO2-), the oxidative end product of NO, directly correlates with intracellular killing of Leishmania major, an obligate intracellular protozoan parasite of macrophages: the level of NO2- production is a quantitative index for macrophage activation. The competitive inhibitor of NO synthesis, monomethylarginine (NGMMLA), inhibits both parasite killing and NO2- production. For Leishmania, the parasite itself participates in the regulation of this toxic effector mechanism. This participation is mediated by parasite induction of tumor necrosis factor alpha (TNF alpha), an autocrine factor of macrophages: NO synthesis by interferon-gamma (IFN-gamma)-treated cells can be blocked by monoclonal antibodies to TNF alpha. NO production by IFN gamma-treated hepatocytes is of special interest in malaria infections: sporozoite-infected hepatocytes kill the intracellular malaria parasite after treatment with IFN gamma; this killing is inhibited by NGMMLA.

Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens.

To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

Passive protection of mice against lethal Francisella tularensis (live tularemia vaccine strain) infection by the sera of human recipients of the live tularemia vaccine.

The relative role that humoral immunity plays in protection against infection with the intracellular bacterium, Francisella tularensis, remains controversial. Cellular immunity is thought to play the major and perhaps only role. The authors, in this article, investigate the immunologic and protective properties of immune serum collected from human recipients of the live tularemia vaccine (LVS). Sera of recipients of the vaccine demonstrated reactivity with the vaccine strain by enzyme-linked immunosorbent assay and Western blot analysis. This reactivity appeared to be directed primarily against the lipopolysaccharide of LVS and demonstrated complete cross-reactivity with fully virulent F. tularensis (Schu4). Pooled immune sera protected mice fully against a 10,000 LD50 challenge with the LVS strain relative to non-immune sera. The protection was abrogated by dilution or preadsorption with the LVS strain but not by preadsorption with Escherichia coli, which suggests specificity of protection. The authors conclude that antibodies to the LVS strain of F. tularensis are generated by live vaccination in humans and play a significant role in protection of mice against lethal challenge with the same organism. These antibodies crossreact completely with fully virulent F. tularensis, but whether they play a role in protection against fully virulent human tularemia strains requires further experimentation.

Lymphokine-induced macrophage resistance to infection with Leishmania major.

Resistance to infection is an effector activity of macrophages that is induced by the cooperation of several molecularly distinct factors in LK: IFN and another nonIFN macrophage activation factor. Unlike many other effector activities of activated macrophages, signal sequence is not critical for induction of resistance to infection. Nor is the activation of macrophages for resistance to infection dependent upon the presence of T lymphocytes in the culture vessel: T cell-depleted peritoneal cell cultures and bone marrow-derived macrophages both develop resistance to infection with L. major in the presence of LK that contains IFN. Further characterization of this activity of activated macrophages will include the identification of LK that cooperate with IFN for induction of resistance to infection, and characterization of the LK-induced changes in macrophage function that mediate this resistant state.

Lymphokine regulation of macrophage effector activities.

Our concept of the regulation of macrophage activation is ever expanding and contracting. In regard to the number of LK that regulate macrophages killing activities, we have entered a new phase. In the beginning there was one macrophage activation factor, MIF; then there were many macrophage activation factors, most uncharacterized and bearing a variety of names. Then came IFN, a genetically cloned single reagent that induced destruction of virtually every target assessed; all activities of macrophages were assumed to be regulated by IFN. Once again, however, the LK universe is expanding: the number of single, cloned reagents that induce macrophage killing activities is amazing. With just two targets, a fibrosarcoma cell and an intracellular amastigote of L. major, we can identify 5 different macrophage activation factors, four of which are cloned and sequenced. As more recombinant reagents become available, the story of macrophage activation is likely to become even more complex. It is fascinating not only that certain of the LK are capable of inducing single effector reactions in the absence of effects on other effector activities, but also that at least one effector reaction requires the cooperation of several molecularly distinct LK. The complexity of LK activation factors that regulate a single effector reaction in vitro is compounded by the complexity in effector cell populations. For example, inflammatory macrophages exposed to LK kill the fibrosarcoma tumor target 5 to 10-fold better than an equal number of resident peritoneal macrophages. In contrast, LK treated resident macrophages eliminate intracellular amastigotes of leishmania far more efficiently than inflammatory cells. Thus, changes in cell populations dramatically affect the capacity to demonstrate a single effector reaction. Further, simple changes in assay conditions also determine whether an effector reaction can be observed in vitro. And superimposed upon all these layers of complexity is the target itself. The mechanisms a macrophages uses to block the replication of a virus may be totally ineffective in the destruction of a multicellular helminth, such as Schistosoma mansoni. And there is no reason to suspect that the extracellular destruction of a tumor target occurs by the same means that the macrophage uses to kill an intracytoplasmic bacterium, such as a rickettsia.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophage activation to kill Leishmania tropica: characterization of a T cell-derived factor that suppresses lymphokine-induced intracellular destruction of amastigotes.

Factors obtained from phorbol myristate acetate (PMA)-stimulated EL-4 thymoma cells, a continuous T cell line, suppressed lymphokine-induced macrophage activation to kill intracellular Leishmania tropica amastigotes. Suppression of this macrophage effector activity was dependent upon concentration of EL-4 fluids admixed with lymphokines in infected macrophage cultures, and was not due to residual PMA or factors released from unstimulated EL-4 cells. Fluids from PMA-stimulated EL-4 cells did not affect the expression of microbicidal activity by macrophages activated in vivo as a consequence of infections with Mycobacterium bovis strain BCG, nor did they abrogate intracellular killing activities by C3H/HeJ macrophages primed by BCG infection and triggered by lymphokines in vitro. That the action of this EL-4 suppressor activity was at the priming stage of macrophage activation was confirmed by kinetic studies: EL-4 fluids added to lymphokine-treated cells in the first 4 hr of treatment completely suppressed intracellular killing of L. tropica; fluids added after 4 hr were not effective. The effects of these EL-4 factors appeared to be selective: of three effector activities of activated macrophages tested, induction of resistance to infection, tumor cytotoxicity, and intracellular destruction of L. tropica, only intracellular killing by lymphokine-treated macrophages was significantly suppressed. These T cell-derived soluble suppressor factor(s) may provide insight into mechanisms of immunosuppression during leishmanial disease and perhaps other intracellular parasitic infections.

Hormonal modulation of sex differences in resistance to Leishmania major systemic infections.

Differences in susceptibility to intravenously inoculated Leishmania major were observed in male and female mice of the BALB/cAnPt, DBA/2N, and DBA/2J strains and (BALB/cAnPt x DBA/2N)F1 hybrids. In all cases, males had significantly higher liver parasite burdens than females. Orchidectomy of BALB/c males resulted in a 20% decrease in the number of parasites in the liver compared with either normal or sham-gonadectomized controls. Additionally, testosterone treatment of female BALB/c mice resulted in an 88% increase in the number of liver amastigotes. These results suggest that the hormone testosterone can modulate systemic L. major infections in BALB/c mice.