Immunological basis for susceptibility and resistance to pulmonary blastomycosis in mouse strains

The immunological basis for a >10-fold resistance of outbred CD-1 mice compared to inbred BALB/c mice to pulmonary blastomycosis was investigated. Bronchoalveolar macrophages (BAM) from CD-1 mice killed yeast cells of Blastomyces dermatitidis (Bd) by 25% and this increased to 59% when activated by IFN-gamma. In contrast, BAM from BALB/c mice lacked significant killing (5%) of Bd but could be activated by IFN-gamma for enhanced killing (19%). Peritoneal macrophages (PM) from CD-1 mice had significant fungicidal activity for Bd (43%) and this increased to 63% with IFN-gamma treatment. By contrast, PM from BALB/c mice did not significantly kill Bd (14%) but were activated by IFN-gamma for significant killing (24%). Fungicidal activity of peripheral blood polymorphonuclear neutrophils (PMN) from CD-1 (87%) was greater than that of BALB/c (75%) (P<0.05). Macrophage inflammatory protein-1alpha (MIP-1alpha) production by BAM from BALB/c was significantly less than that from CD-1 in response to co-culture with Bd. IFN-gamma production by CD-1 spleen cells in response to concanavalin A (Con A, 1microg/ml) was 8-fold greater than that by BALB/c spleen cells. In contrast, BAM and PM from BALB/c mice in co-culture with Bd secreted several-fold more TNFalpha than BAM or PM from CD-1 mice. IL-2 production by BALB/c spleen cells in response to Con A was 3- to 4-fold greater than that by CD-1 spleen cells. Depressed IL-2 production by Con A stimulated CD-1 spleen cells correlated with depressed proliferative responses. Resistance of CD-1 mice to pulmonary blastomycosis correlates with enhanced fungicidal activity of BAM, PM, PMN, and IFN-gamma production by Con A stimulated spleen cells, compared to BALB/c mice. Consistent with the in vitro enhancement of effector cell function by IFN-gamma, in vivo therapy with IFN-gamma significantly (P<0.0001) improved survival of BALB/c mice with pulmonary blastomycosis.     

Role of activated macrophages in Acanthamoeba keratitis

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-gamma) -containing supernatants were collected 24 hr later. The IFN-gamma-containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A-activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A-stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A-stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A-treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-gamma-containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin D, but not by L-N6-1-iminoethyl-L-lysine dihydrochloride (L-NIL), which is a selective inhibitor of inducible NO synthase (INOS).     

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.     

Effect of a local immune reaction on peripheral blood polymorphonuclear neutrophil microbicidal function: studies with fungal targets

Peripheral blood polymorphonuclear neutrophils (PMN) from mice immunized with Blastomyces dermatitidis and then stimulated locally (intraperitoneally, ip) with B. dermatitidis antigen had enhanced killing of B. dermatitidis in vitro (54.4 +/- 19.49 of inoculum) compared to nonimmune mice (32.7 +/- 8.7%; P less than 0.02), nonimmune mice given antigen ip (30.6 +/- 14.0%; P less than 0.05), or immune mice not given antigen ip (15.4 +/- 9.9%; P less than 0.01). Peripheral blood PMN from all four groups had marked killing ability against Candida albicans (91.8-99.3% of inoculum). That the killing of B. dermatitidis was due to PMNs was demonstrated by lack of killing by isolated peripheral blood mononuclear cells from all four groups. A local immune reaction can result in enhancement of PMN fungicidal activity, and this is reflected even in peripheral blood PMN. We hypothesize this is an important component of normal host defenses against fungal infection, and likely other microbial infections. Enhancement of PMN microbicidal function by the soluble mediators presumed to be responsible for the effects observed may be an approach to immunomodulating therapy or prophylaxis of infection.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). II. Kinetics of the production and characterization of the producer

Kinetics of the production of a stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF) (1) from Con A-stimulated SD rat spleen cells, and the cells involved in the production of the factor, were examined comparatively with those of interleukin 2 (IL 2) and interferon (IFN). STIF activity in the culture supernatants reached a plateau 24 hr after the culture, and the plateau level was maintained during an additional culture for 72 hr. Characterization of the cells involved in STIF production by means of negative selection of unfractionated or nylon-fractionated spleen cells with anti-rat T cell serum or monoclonal antibodies plus complement revealed that the cells are nylon-nonadherent T cells bearing a suppressor T cell marker. The nylon-nonadherent T cell population did not require additional macrophages for STIF production. In vivo pretreatment of rats with cyclophosphamide (25 to 100 mg/kg) reduced or abolished the production of STIF from the Con A-stimulated spleen cells of the rats. The STIF production from Con A-stimulated spleen cells was inhibited by the addition of indomethacin at 10 ng/ml, indicating a regulatory role of prostaglandin(s) in STIF production. The above characteristics distinguish a T cell subset for STIF production from those for IL 2 and IFN production.     

In vitro killing of schistosomula of Schistosoma mansoni by BCG and C. parvum-activated macrophages.

Resistance to Schistosoma mansoni infection in the mouse has been induced either specifically by a primary infection with this parasite or nonspecifically by a variety of immunostimulants such as BCG. In the present study we developed an in vitro system to examine the effector mechanism of nonspecifically induced resistance. Activated macrophage monolayers obtained from BCG- or Corynebacterium parvum treated mice killed a respective mean 32 +/- 6% and 48 +/- 5% of schistosomula after 24 hr incubation. The killing of the parasites was verified by their inability to mature to adult worms upon injection into normal mice. The activated macrophage-mediated killing was related to cell:parasite ratio, and was partially lost if the macrophage monolayers were kept in cultures for 24 hr before incubation with the organism. Supernatants of macrophages cultured in the presence of schistosomula killed a mean of 51 +/- 3% of the organisms whereas those from cells cultured alone resulted in a mean killing of 25 +/- 3%. Furthermore, toxic supernatants could be generated equally well on incubation with S. mansoni schistosomula or Trichinella spiralis larvae. Our data show that activated macrophage monolayers through soluble mediators destroy a significant proportion of the multicellular parasite S. mansoni schistosomula in vitro.     

IL-5-Induced Eosinophils Suppress the Growth of Leishmania amazonensis In Vivo and Kill Promastigotes In Vitro in Response to Either IL-4 or IFN-gamma

In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

Opioid peptides modulate production of interferon gamma by human mononuclear cells

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.     

Kinetics and requirements for activation of macrophages for fungicidal activity: effect of protein synthesis inhibitors and immunosuppressants on activation and fungicidal mechanism.

Peritoneal-and pulmonary macrophages can be activated in vitro with lymphokines (LK) or IFN-gamma, without exogenous lipopolysaccharide, for fungicidal activity against several pathogenic fungi. However, neither the biochemical nor metabolic events of the activation process or of the effector phase have been defined. In the present work we sought to elucidate these events with time-course studies using inhibitors of protein synthesis as well as immunosuppressive agents. We found that protein synthesis inhibitors abrogated the activation process, because cycloheximide (CHX) (1-2 micrograms/ml) prevented activation of macrophages for fungicidal activity against Candida albicans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. Blocking of the activation process by CHX was not due to macrophage cytotoxicity, and CHX did not impair the ability of nonactivated macrophages to kill Candida parapsilosis. In kinetic studies we showed that activation of macrophages was induced in 4 hr of LK treatment and that CHX had no effect if added after this time. In contrast to CHX, therapeutic concentrations of hydrocortisone (HC), such as less than or equal to 5 micrograms/ml, or cyclosporin A (CsA), 5 micrograms/ml, did not significantly inhibit LK activation of macrophages for killing of fungi. In the effector phase, the fungicidal capacity of activated macrophages in short-term (less than or equal to 4 hr) killing assays could not be abrogated by CHX (5 micrograms/ml), HC (100 micrograms/ml), or CsA (10 micrograms/ml). These results demonstrate that the activation but not the effector mechanism of macrophages for fungicidal activity is blocked by inhibition of protein synthesis. In contrast, therapeutic concentrations of HC or CsA may not interfere with activation of macrophages or their killing mechanisms, thus providing a rationale for antifungal immunotherapy in certain clinical situations (e.g., infection in the immunosuppressed patient).     

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.     

Interferon and natural killing of human lymphoma cell lines after induction of the Epstein Barr viral cycle by superinfection

Interferon (IFN) production during natural killer (NK) cell assays with Raji, an EBV-carrying human lymphoma-derived cell line, was studied to determine whether IFN generated by effectors in vitro acted in target cell lysis. In 4-hr tests, Raji is insensitive to NK but becomes susceptible after superinfection with the P3HR-1 strain of EBV. IFN was not detectable by bioassay in supernatants from 4-hr assays, and the addition of antibody to IFN did not prevent the lysis of the superinfected Raji cells. In 18-hr tests the NK sensitivity of the superinfected Raji cells was markedly elevated, and a percent of the normal Raji cells was also killed. IFN alpha was found in supernatants from 18-hr tests. Antibody to IFN alpha markedly reduced the killing of superinfected Raji and slightly reduced cytotoxicity against control Raji in 18-hr tests. Taken together these results indicate that what is referred to as natural killing has IFN-related and IFN-nonrelated components.     

Production of and responsiveness to interleukin 2 in autoimmune BXSB mice

BXSB male mice serve as one of several murine models of human systemic lupus erythematosus. T-cell abnormalities in these mice involve decreased production of and responsiveness interleukin 2 (IL-2) and are age-related. The studies presented here investigated the mechanism of these T-cell defects. The results suggest that excessive suppressor-T-cell activity as well as soluble inhibitors of IL-2 production and activity, including PGE, are not responsible for the low levels of IL-2 observed in culture supernatants of Con A-stimulated lymphocytes from "old" (3-6 months) BXSB male mice. Supplementation of Con A-stimulated lymphocyte cultures from BXSB male mice with human IL-1 or normal murine accessory cells did not augment IL-2 production. Reduced proliferative responses were observed in bulk cultures of Con A- or alloantigen-stimulated "old" BXSB male lymphocytes, which were not enhanced by exogenous IL-2. Limiting dilution analysis revealed reduced frequencies of Con A- and alloantigen-inducible IL-2-reactive T cells in these mice. These results suggest intrinsic defects in the ability of T cells from "old" BXSB male mice to be activated to produce and respond to IL-2.     

Effect of concanavalin A treatment on the allogeneic response of mice to challenge with P 815 mastocytoma: interleukin 2 treatment reverses concanavalin A suppression in vivo

Mice injected repeatedly with concanavalin A (Con A) prior to and following challenge with P 815 mastocytoma are suppressed in their cell-mediated cytotoxicity responses. Earlier studies showed that pretreatment of the animals with silica to affect macrophage (M phi) functions reversed the Con A suppression. In the present paper we have shown that peritoneal exudate cells (PEC) induced/activated by ip injection of Con A were able to transfer suppression to normal mice. Separation of the PEC populations into adherent and nonadherent cells abrogated their capacity to transfer suppression. It was further shown that Con A is not functioning in this in vivo system to block effector activity of cytotoxic cells on target cells, and PEC induced with Con A were not directly cytotoxic to target P 815 cells. Finally, we were able to show that the cytotoxicity response of Con A-suppressed mice could be enhanced by treatment with concentrated culture supernatants of normal mouse spleen cells, rich in interleukin 2 (IL 2) activity. Attempts to detect a recently described mouse serum inhibitor of IL 2 in normal or Con A-treated mice were unsuccessful and spleen cells from Con A-treated mice lost their capacity to generate IL 2 in vitro when cultured under appropriate conditions. Taken together, these results suggest that suppression of cell-mediated immune responses in Con A-treated mice results from interruption of the normal generation of IL 2 helper effects necessary for the activation of cytotoxic effector T cells in vivo.     

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.     

Prostaglandin E2 inhibits Paracoccidioides brasiliensis killing by human monocytes

Human monocytes lacked fungicidal activity against high virulence strain of Paracoccidioides brasiliensis, even after IFN-gamma activation. However, monocytes treated with indomethacin exhibited an effective killing against this fungus, suggesting a role of prostaglandin E2 (PGE2) in the inhibition process. Thus, the purpose of this work was to determine whether the effect of PGE2 in fungicidal activity was related with decrease on H(2)O(2) release, the metabolite involved in P. brasiliensis killing, and changes in the levels of TNF-alpha, IL-6 and IL-10. Human monocytes challenged with the fungus produced high PGE2 levels, which in turn inhibited the fungicidal activity of these cells by reducing H(2)O(2) and TNF-alpha production.     

Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine

Peritoneal macrophages from CBA mice incubated with rIFN-gamma are effective in killing the protozoal parasite Leishmania major in vitro. This leishmanicidal activity can be completely inhibited by L-NG-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine:nitric oxide (NO) pathway. The culture supernatants of macrophage activated by IFN-gamma contain increased levels of NO2-, the production of which is inhibited by L-NMMA, but not by its D-enantiomer. L. major promastigotes are killed when incubated at room temperature in PBS containing NO. These data demonstrate that NO is an effector mechanism in macrophage killing of intracellular protozoa. The importance of NO in vivo is demonstrated by the finding that CBA mice infected with L. major developed exacerbated disease when L-NMMA was injected into the lesions, resulting in 10(4)-fold increases in the number of parasites extractable from the lesions.     

Recombinant mouse gamma interferon induces the priming step in macrophage activation for tumor cell killing

Mouse macrophages become activated to kill tumor cells by traversing a series of steps (1-3). The first of these does not cause the expression of cytolytic activity; instead, it primes macrophages to respond to a second signal(s) that then triggers the onset of killing (4-7). The mediator that is responsible for priming is contained, along with other lymphokines, in the culture supernatants of concanavalin A-stimulated spleen cells (5-7) cloned T lymphocytes (8), or some T cell hybridomas (9). Close association has been noted between macrophage priming activity and antiviral activity that is attributable to gamma interferon (10-12). However, unequivocal evidence that the two activities are products of the same molecule has not been available. We now how conclusively by using mouse gamma interferon (MulFN-gamma)3 produced by recombinant DNA technology that, in addition to antiviral activity, this lymphokine has the capacity to induce the priming step in the process of macrophage activation for tumor cell killing.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

Resistance to macrophage-mediated killing as a factor influencing the pathogenesis of chronic cutaneous leishmaniasis

Cutaneous leishmaniasis can be either a spontaneously healing or chronic disease, depending upon the strain of parasite and the immunological status of the host. We have investigated parasite factors responsible for the variable pathogenesis observed in leishmanial infections by testing the sensitivity of several leishmanial strains to intracellular killing in lymphokine (LK) activated mouse macrophages. Significant microbicidal activity against Leishmania tropica, a strain which heals in C57BL/6 (B6) mice, was found. In contrast, a strain (Maria) which has previously been shown to induce chronic nonhealing cutaneous lesions in B6 mice was resistant to killing in activated macrophages. This resistance to killing was observed in macrophages activated by LK obtained from either Bacille Calmette-Guérin-, L. tropica, or the Maria strain infected mice. The inability of LK activated macrophages to kill the Maria strain was shown not to be due to parasite induced inhibition of killing mechanisms, since Maria strain infected, LK treated macrophages exhibited tumoricidial activity similar to uninfected macrophages. Furthermore, LK activated macrophages simultaneously infected with the Maria strain and another intracellular pathogen, Toxoplasma gondii, killed Toxoplasma, but not the Maria strain. Temperature was also found to significantly influence the multiplication and killing of Leishmania parasites. As would be expected from their cutaneous nature, L. tropica and Maria strain parasites multiplied better at 35 degrees C than at 37 degrees C. Also consistent with the failure of cutaneous strains to visceralize in immunocompetent mice was the observation that the killing of leishmanial parasites was enhanced at the higher temperature. Thus, the temperature dependent growth capacity and sensitivity to killing of a given leishmanial strain in macrophages may be important factors influencing the pathogenesis of cutaneous leishmaniasis.