Detection of active tuberculosis by an MPB-64 transdermal patch: a field study.

The mycobacterial antigen MPB-64 was formulated for delivery in a transdermal patch and used as a diagnostic skin test reagent to detect active tuberculosis (TB) in patients attending a clinic in Manila, The Philippines. The MPB-64 Transdermal Patch was applied to 62 patients, 49 with sputum-positive active disease and 13 who had completed TB chemotherapy, and to 28 non-TB but tuberculin-positive controls. The results were read at 72 h. The sensitivity of the Transdermal Patch was 87.8%, with an efficacy of 92.9% and a specificity of 100%. The 13 TB patients who had completed 6 months of TB chemotherapy showed different reactions to the MPB64 patch test: those who had completed chemotherapy < 4 months before testing were positive; 50% of patients who completed chemotherapy 5 months previously were positive; and those who had completed chemotherapy 7 and 8 months before were negative. All the non-TB controls with positive tuberculin tests were negative to the MPB-64 Transdermal Patch, even at the highest protein dose tested. This test may be a useful method to distinguish active TB patients from TB-infected but asymptomatic individuals. Moreover, the MPB64 Transdermal Patch may be useful to monitor successful chemotherapy.

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.

Interaction of anti-cholesterol antibodies with human lipoproteins.

Inoculation of mice with cholesterol-rich liposomes containing the adjuvant monophosphoryl lipid A results in the production of antiserum containing IgM Ab to cholesterol. The specificity of the Ab was to cholesterol and structurally similar sterols containing a 3 beta-hydroxyl group. Anti-cholesterol binding activity was significantly diminished if the 3 beta-hydroxyl was altered by either epimerization, substitution, oxidation, or esterification. A similar specificity for 3 beta-hydroxy-sterols was observed for an anti-cholesterol IgM mAb. Both hyperimmune serum and the mAb reacted with intact human very-low-/intermediate-density lipoprotein (VLDL/IDL) and low-density lipoproteins (LDL), but not high-density lipoproteins (HDL), in an ELISA, but could react with total lipid extracts containing cholesterol that were prepared from all three lipoprotein classes. Functionally, immune serum or the mAb aggregated and induced a fusion-like reaction with VLDL/IDL and LDL at low temperatures: these aggregates result in spherical structures visible with light microscopy. Similarly, binding of anti-cholesterol A to small cholesterol-rich liposomes resulted in the appearance of vesicular structures with approximately 20- to 200-fold increased diameters. These data demonstrate that the anti-cholesterol Ab recognize unesterified cholesterol in VLDL/IDL and LDL; high-density lipoprotein cholesterol in the intact lipoprotein, however, appears to be protected from reaction with these Ab.

Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth.

Murine macrophages supported exponential intracellular growth of Francisella tularensis LVS in vitro with a doubling time of 4 to 6 h. LVS was internalized and remained in a vacuolar compartment throughout its growth cycle. The importance of endosome acidification to intracellular growth of this bacterium was assessed by treatment of LVS-infected macrophages with several different lysosomotropic agents (chloroquine, NH4Cl, and ouabain). Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidification no longer supported replication of LVS. Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered from treated macrophage cultures was equivalent to the input inoculum (approximately 10(4) CFU) at 72 h. In contrast, over 10(8) CFU was consistently recovered from untreated cultures. Pretreatment of macrophages with these endosome acidification inhibitors did not alter their ingestion of bacteria. Further, the effects of the inhibitors were completely reversible: inhibitor-pretreated LVS-infected macrophages washed free of the agent and cultured in medium fully supported LVS growth over 72 h. Endosome acidification is an important cellular event essential for release of iron from transferrin. The growth-inhibitory effects of both chloroquine and NH4Cl were completely reversed by addition of ferric PPi, a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin. Thus, intracellular localization in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacterium, and iron depletion is one mechanism that macrophages use to inhibit its growth.

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.

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.

Macrophage activation: a riddle of immunological resistance.

Various lines of defense against infection are present in all living creatures. The balance between symbiosis and parasitism is determined by the mechanisms through which the host resists infection and by the extent of injury induced by the parasite: both factors contribute to disease. Lines of host defense can be arbitrarily divided into three components: 1) barrier functions of skin and mucous membranes and their innate physical and secretory antimicrobial components; 2) elements of host defense that do not necessarily require prior exposure to an infectious agent or immunologic memory (mast cells, granulocytes, macrophages, NK cells, gamma/delta T cells); and 3) immune responses directed against specific epitopes on the infectious agent induced by prior exposure and immunologic memory (alpha/beta T cells, B cells). Analysis of such host defense mechanisms repeatedly documents tremendous redundancy and overlap between these lines of defense. Further, there is open communication, so that a change at any one level ripples throughout the system. Acquired nonspecific resistance to infection is an example of such a ripple. Host response to one infection alerts the immune system, so that the general level of resistance to other infectious agents is increased. This response is initiated by an immune response (third line of defense) but effected by nonspecific elements (second line of defense). The survival value of such responses is obvious. There are numerous examples in both mouse and man of the operation of these systems in response to infection. Further, the menus of antimicrobial components available to both mouse and man for resistance to infection are very similar, but not identical. Indeed, it is said that the genetic basis for differences between mice and man revolve around a difference of less than 10% in DNA sequences. But there are differences! Mouse macrophages produce IFN-beta in response to infection, human cells produce IFN-alpha. Mouse macrophages effect antimicrobial activity principally through induction of NO synthase and the generation of toxic nitrogen oxides. This pathway has yet to be described with human macrophages. In both man and mouse, F. tularensis is an obligate intracellular parasite of macrophages that requires an essential component provided by the cell for its replication. That mouse and man are not so different is well illustrated by the effector mechanisms induced by IFN-gamma for antimicrobial activity against F. tularensis.(ABSTRACT TRUNCATED AT 400 WORDS)

IFN-gamma produced in vivo during the first two days is critical for resolution of murine Leishmania major infections.

Resolution of murine infection with Leishmania major is dependent upon the presence of IFN-gamma during the first week of infection. To more precisely determine the period during which IFN-gamma is critical, we infected footpads of resistant C3H/HeN mice with amastigotes of L. major and treated these mice with neutralizing monoclonal Ab (MAb) specific for IFN-gamma on successive days. Mice treated with anti-IFN-gamma MAb on or before day 2 had significantly enlarged lesions, and increased parasites in lesions, compared with mice treated with an isotype control MAb. In contrast, mice treated with anti-IFN-gamma MAb on day 3 or later resolved their lesions and had no parasites at the inoculation site. Related experiments obtained with a neutralizing MAb specific for TNF-alpha demonstrated the critical role TNF-alpha plays in resolution of Leishmania infection. Thus, IFN-gamma and TNF-alpha were both critical for resolution of infection with L. major, with IFN-gamma's role limited to the first 2 days.

T-cell-independent resistance to infection and generation of immunity to Francisella tularensis.

The intraperitoneal 50% lethal dose (LD50) for Francisella tularensis LVS in both normal control heterozygote BALB/c nu/+ mice and BALB/c nu/nu mice was 2 x 10(0). Both nu/+ and nu/nu mice given 10(7) LVS bacteria or more intradermally (i.d.) died, with a mean time to death of about 7 to 8 days. On the other hand, nu/+ mice given 10(6) LVS bacteria or less survived for more than 60 days and cleared systemic bacteria, while nu/nu mice given 10(6) LVS bacteria or less survived for more than 10 days but died between days 25 and 30. Thus, the short-term (i.e., < 10-day) i.d. LD50 of both nu/nu and nu/+ mice was 3 x 10(6), but the long-term (i.e., > 10-day) i.d. LD50 of nu/nu mice was less than 7 x 10(0). The short-term survival of i.d. infection was dependent on tumor necrosis factor and gamma interferon: treatment of nu/nu mice with anti-tumor necrosis factor or anti-gamma interferon at the time of i.d. infection resulted in death from infection 7 to 8 days later, whereas control infected nu/nu mice survived for 26 days. nu/nu mice infected with LVS i.d. generated LVS-specific serum antibodies, which were predominantly immunoglobulin M: titers peaked 7 days after i.d. infection but declined sharply by day 21, after which mice died. Surprisingly, nu/nu mice given 10(3) LVS bacteria i.d. became resistant to a lethal challenge (5,000 LD50s) of LVS intraperitoneally within 2 days after i.d. infection; nu/nu mice similarly infected with LVS i.d. and challenged with Salmonella typhimurium (10 LD50s) were not protected. nu/nu mice given nu/+ spleen cells intravenously as a source of mature T cells survived i.d. infection for more than 60 days and cleared bacteria. Taken together, these studies demonstrate that i.d. infection of nu/nu mice with LVS rapidly generates T-cell-independent, short-term, specific protective immunity against lethal challenge, but T lymphocytes are essential for long-term survival.

Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice.

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.

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.

Detection of Francisella tularensis in blood by polymerase chain reaction.

We developed a polymerase chain reaction-based assay for Francisella tularensis which we evaluated by using spiked blood samples and experimentally infected mice. The assay detected both type A and type B F. tularensis at levels equivalent to one CFU/microliter of spiked blood. Results from polymerase chain reaction-based assay of limiting dilutions of blood from mice infected with the live vaccine strain agreed closely with results from blood culture.

Introduction of Francisella tularensis at skin sites induces resistance to infection and generation of protective immunity.

Mice are susceptible to systemic infection with Francisella tularensis strain LVS; thus, the intraperitoneal (i.p.) lethal dose at 50% (LD50) in C3H/HeN and C57BI/6J mice is only a single bacterium, while the intradermal (i.d.) LD50 is more than 10(4). Here we show that the LD50 when LVS is introduced via the skin, either i.d. or subcutaneously (s.c.), ranges from 7 x 10(4) to 2 x 10(6). Sublethal i.d. or s.c. infection (priming) invariably leads to the generation of systemic and specific protective immunity: primed mice survive lethal i.p., intravenous (i.v.), or i.d. challenges of LVS but not Salmonella typhimurium W118 or Escherichia coli 018:K1:H7 strain BORT.

Rapid generation of specific protective immunity to Francisella tularensis.

Mice inoculated either subcutaneously (s.c.) or intradermally (i.d.) with a sublethal dose of Francisella tularensis LVS are immune to a lethal intraperitoneal (i.p.) or intravenous (i.v.) challenge of LVS. Here, we show that this immunity developed quite rapidly: mice given a sublethal dose of live LVS s.c. or i.d. (but not i.v.) withstood lethal i.p., i.v., or i.d. challenge as early as 2 days after the initial inoculation, despite the presence of bacterial burdens already in tissues. The magnitude of this early protection was quite impressive. The i.p. 50% lethal dose (LD50) in naive C3H/HeN mice was only 2 bacteria, while the i.p. LD50 in mice given 10(4) LVS i.d. 3 days previously was 3 x 10(6) bacteria. Similarly, the i.v. LD50 in C3H/HeN mice shifted from 3 x 10(2) in naive mice to 5 x 10(6) in primed mice within 3 days after i.d. LVS infection. Comparable changes in the i.p. and i.v. LD50 were observed in C57BL/6J mice. This rapid generation of protective immunity was specific for LVS, in that mice given a sublethal i.d. inoculation of LVS did not survive a lethal challenge with either Salmonella typhimurium W118 or Escherichia coli O118 BORT at any time, nor could mice given sublethal doses of S. typhimurium, E. coli, or Mycobacterium bovis BCG survive lethal doses of LVS. Although an increase in the mean time to death from S. typhimurium infection was noted when mice were given a sublethal i.d. dose of LVS 4 to 14 days earlier, no overall increase in protection or change in the S. typhimurium LD50 was observed. Thus, sublethal infection with LVS at skin sites induced rapid and specific protective immunity.

Cytotoxic activity and production of toxic nitrogen oxides by macrophages treated with IFN-gamma and monoclonal antibodies against the 73-kDa lipopolysaccharide receptor.

The hamster IgM mAb 5D3 is specific for an 73-kDa LPS receptor on murine leukocytes. This mAb inhibits binding of radiolabeled LPS to splenocytes and acts as an agonist for induction of LPS-mediated changes in macrophage function. Resident peritoneal macrophages treated with IFN-gamma and mAb 5D3 developed potent cytotoxic activity against tumor cells. Cells treated with IFN-gamma or mAb 5D3 alone were inactive. Macrophage cytotoxic activity induced by IFN-gamma and mAb 5D3 was inhibited by NGMMLA and coincident with high levels of NO2-released into culture fluids. These data show that mAb 5D3 serves as an effective trigger signal for induction of cytotoxic activity with IFN-gamma-primed macrophages. Indeed, mAb 5D3 exactly mimicked the effects of LPS in these same systems. Unlike LPS, effects of mAb 5D3 on induction of macrophage cytotoxic activity and production of nitrogen oxides was abrogated after boiling, and not affected by addition of polymyxin B. The effects of LPS and mAb 5D3 as a trigger signal for IFN-gamma-primed macrophages were associated with production of TNF activity in culture fluids and inhibited by mAb against rTNF-alpha. Expression of class II MHC on macrophages induced by IFN-gamma treatment was suppressed by both LPS and mAb 5D3. These suppressive effects of LPS and mAb 5D3 were not affected by NGMMLA or mAb against rTNF-alpha. Finally, macrophages treated with LPS or mAb 5D3 before exposure to IFN-gamma and LPS or mAb 5D3 did not develop cytotoxic activity or high levels of NO2- in the culture fluids. These same cells developed both effector activities after addition of rTNF-alpha. These results in toto identify the 73-kDa protein as a receptor that mediates LPS-induced changes in macrophage effector function. The mAb 5D3 serves as a specific and defined reagent agonist for analysis of LPS receptor-linked change.

Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules.

Francisella tularensis live vaccine strain (LVS) was grown in culture with nonadherent resident, starch-elicited, or Proteose Peptone-elicited peritoneal cells. Numbers of bacteria increased 4 logs over the input inoculum in 48 to 72 h. Growth rates were faster in inflammatory cells than in resident cells: generation times for the bacterium were 3 h in inflammatory cells and 6 h in resident macrophages. LVS-infected macrophage cultures treated with lymphokines did not support growth of the bacterium, although lymphokines alone had no inhibitory effects on replication of LVS in culture medium devoid of cells. Removal of gamma interferon (IFN-gamma) by immunoaffinity precipitation rendered lymphokines ineffective for induction of macrophage anti-LVS activity, and recombinant IFN-gamma stimulated both resident and inflammatory macrophage populations to inhibit LVS growth in vitro. Inflammatory macrophages were more sensitive to effects of IFN-gamma: half-maximal activity was achieved at 5 U/ml for inflammatory macrophages and 20 U/ml for resident macrophages. IFN-gamma-induced anti-LVS activity correlated with the production of nitrite (NO2-), an oxidative end product of L-arginine-derived nitric oxide (NO). Anti-LVS activity and nitrite production were both completely inhibited by the addition of either the L-arginine analog NG-monomethyl-L-arginine or anti-tumor necrosis factor antibodies to activated macrophage cultures. Thus, macrophages can be activated by IFN-gamma to suppress the growth of F. tularensis by generation of toxic levels of NO, and inflammatory macrophages are substantially more sensitive to activation activities of IFN-gamma for this effector reaction than are more differentiated resident cells.

In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies.

The role(s) of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) in establishment and maintenance of protective immunity to Francisella tularensis LVS in mice (C3H/HeN) was examined by selective removal of these cytokines in vivo with neutralizing antibodies. The 50% lethal dose (LD50) for mice infected intradermally with F. tularensis alone was 136,000 CFU; treatment of mice with anti-IFN-gamma or anti-TNF-alpha at the time of infection significantly reduced (P much less than 0.05) the LD50 to 2 and 5 CFU, respectively. Abrogation of protective immunity, however, was effective only when anti-IFN-gamma or anti-TNF-alpha was administered prior to day 3 postinfection. In contrast, the LD50 for mice treated with anti-IL-4 was repeatedly higher (555,000 CFU) than for controls; this difference, however, was not significant (P greater than 0.05). Thus, IL-4 may be detrimental, while IFN-gamma and TNF-alpha were clearly crucial to the establishment of protective immunity to F. tularensis during a primary infection. The importance of IFN-gamma and TNF-alpha during a secondary immune response to F. tularensis was also investigated. Spleen cells from immune mice passively transfer protective immunity to recipient mice in the absence of confounding antibody-mediated immunity. This passive transfer of immunity, however, was abrogated by treatment of recipient mice with anti-IFN-gamma or anti-TNF-alpha at the time of challenge infection. That anticytokines effectively abrogate protective immunity very early in the course of infection with F. tularensis suggests that T-cell-dependent activation of macrophages for microbicidal activity is unlikely. These T-cell-independent events early in the course of infection may suppress bacterial replication until a T-cell-dependent response ultimately clears the bacteria.