Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol.

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

Apoptosis driven infection.

Professional phagocytes like polymorphonuclear neutrophil granulocytes (PMN) and macrophages (MF) kill pathogens as the first line of defense. These cells possess numerous effector mechanisms to eliminate a threat at first contact. However, several microorganisms still manage to evade phagocytic killing, survive and retain infectivity. Some pathogens have developed strategies to silently infect their preferred host phagocytes. The best example of an immune silencing phagocytosis process is the uptake of apoptotic cells. Immune responses are suppressed by the recognition of phosphatidylserine (PS) on the outer leaflet of their plasma membrane. Taking Leishmania major as a prototypic intracellular pathogen, we showed that these organisms can use the apoptotic "eat me" signal PS to silently enter PMN. PS-positive and apoptotic parasites, in an altruistic way, enable the intracellular survival of the viable parasites. Subsequently these pathogens again use PS exposition, now on infected PMN, to silently invade their definitive host cells, the MF. In this review, we will focus on L. major evasion strategies and discuss other pathogens and their use of the apoptotic "eat me" signal PS to establish infection.

Interferon-gamma-dependent mechanisms of mycobacteria-induced pulmonary immunopathology: the role of angiostasis and CXCR3-targeted chemokines for granuloma necrosis.

The mechanisms leading to granuloma caseation, a hallmark of tuberculosis (TB) in humans, are poorly understood. Lung histopathology of C57BL/6 (WT) mice 16 weeks after aerosol infection with Mycobacterium avium strain TMC724 is uniquely characterized by centrally necrotizing granulomas, strongly resembling human TB lesions. However, IFN-gamma-deficient (GKO) and IFN-gamma-receptor-deficient (GRKO) mice did not develop granuloma necrosis following M. avium infection. Comparison of differentially expressed genes in infected WT and GKO lungs by DNA microarray and RNase protection assays revealed that the angiostatic chemokines CXCL9-11 were significantly reduced in GKO mice. In contrast, angiogenic mediators such as angiopoietin and vascular endothelial growth factor, and angiogenic chemokines such as CXCL2, CCL3, and CCL4, remained unchanged or were expressed at higher levels than in infected WT mice, suggesting impaired neovascularization of the granuloma as a possible mechanism for caseation in WT mice. Granuloma vascularization was significantly decreased in central, but not peripheral, areas of granulomas of infected WT compared to GKO mice. In contrast to GRKO mice, WT mice showed signs of severe hypoxia in cells immediately surrounding the necrotic core of granulomas as measured immunohistochemically with a reagent detecting pimonidazole adducts. To test the hypothesis that CXCR3, the common receptor for the angiostatic chemokines CXCL9-11, is involved in granuloma caseation, histomorphology was assessed in M. avium-infected mice deficient for CXCR3 (CXCR3-KO). 16 weeks after infection, these mice developed caseating granulomas similar to WT mice. We conclude that IFN-gamma causes a dysbalance between angiostatic and angiogenic mediators and a concomitant reduction in granuloma vascularization, but that CXCR3-targeted chemokines are not sufficient to induce granuloma necrosis in a mouse model of mycobacteria-induced immunopathology.

Different patterns of the L-histidine decarboxylase (HDC) gene expression in mice resistant and susceptible to experimental cutaneous leishmaniasis.

OBJECTIVE AND DESIGN: In the present study the experimental murine Leishmania major ( L. major) infection model was used to investigate the role of histamine biosynthesis in cutaneous leishmaniasis. SUBJECTS, TREATMENT AND METHODS: A novel RNase Protection Assay (RPA) was developed and applied for the assessment of L-histidine decarboxylase (HDC) gene expression in organs of resistant C57BL/6 and susceptible BALB/c mice after infection with L. major. RESULTS: In the acute phase of infection a rapid but transient induction of HDC expression was observed in the infected lymph nodes of both strains correlating both temporally and spatially with parasite spread. The signal was present in the draining popliteal lymph nodes of both hosts, however, only susceptible mice known to be unable to control parasite dissemination showed induction of HDC in their distant periaortic lymph nodes as well. During the chronic phase of infection only the heavily parasitized organs of BALB/c mice showed high HDC gene expression. CONCLUSIONS: These data suggest that expression of the histamine-producing enzyme HDC in the decisive acute phase of leishmaniasis is not coupled with development of either appropriate Th1 or inadequate Th2 responses to L. major. We hypothesize, however, that during the chronic phase of infection elevated HDC levels, possibly of mast cell origin, are associated with Th2-dominated responses and serious disease development.

Leishmania promastigotes release a granulocyte chemotactic factor and induce interleukin-8 release but inhibit gamma interferon-inducible protein 10 production by neutrophil granulocytes.

Recent data from our laboratory suggest that neutrophil granulocytes (polymorphonuclear leukocytes [PMN]) can serve as host cells for Leishmania major in the early phase of infection. In line with these findings, an early influx of PMN to the infected tissues was shown by others to be associated with susceptibility to infection with L. major. The mechanisms underlying the initial PMN recruitment to the site of infection is poorly understood. In the present study we investigated whether Leishmania can influence PMN migration. Supernatants of Leishmania promastigotes were tested for their chemotactic activity using an in vitro chemotaxis assay. All Leishmania species tested (L. major, L. aethiopica, and L. donovani) displayed a marked chemotactic effect on human PMN. However, no effect on the migration of macrophages and NK cells was observed. Checkerboard analysis revealed that the observed PMN migration was due to chemotaxis rather than chemokinesis. Most of the chemotactic activity was found in fractions containing molecules with sizes between 10 and 50 kDa. Pretreatment of PMN with N-formyl-methionyl-leucyl-phenylalanine blocked the chemotactic activity of Leishmania supernatants up to 75%. In addition, we found that leishmanial contact induced the release of interleukin-8 (IL-8) and inhibited the production of gamma interferon-inducible protein 10 (IP-10) by PMN. These data suggest that infection with Leishmania promastigotes leads to PMN accumulation via the production of a chemotactic factor by the parasites, and this effect is amplified by the induction of IL-8 production in PMN. On the other hand, the inhibition of IP-10 production can lead to prevention of NK cell activation.

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes.

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.

Novel multi-probe RNase protection assay (RPA) sets for the detection of murine chemokine gene expression.

Chemokines play an essential role in immune and inflammatory reactions via the recruitment of leukocytes. Studying the role of chemokines in vivo is complicated by the redundancy of their action and by their promiscuous receptor usage. The simultaneous analysis of several chemokines is, therefore, advantageous in order to obtain a comprehensive view of chemokine participation in inflammatory and infectious processes. At present, no multi-probe detection systems are available for the analysis of recently described chemokines. In this study, new multi-probe RNase protection assay (RPA) template sets were developed for the analysis of murine chemokines. Chemokine cDNA fragments were generated by RT-PCR and individually subcloned into the plasmid pGEM-T providing a T7 promotor. In this way, two multi-probe template sets were constructed each containing six chemokine sequences (CXCL12/SDF-1, XCL1/lymphotactin, CCL20/exodus-1, CCL25/TECK, CX3CL1/fractalkine, CXCL1/KC, and CCL20/MDC, CXCL9/MIG, CCL9/10/MIP-1gamma, CXCL13/BLC, CCL12/MCP-5, CCL19/ELC, respectively) and templates for the two house-keeping genes L32 and GAPDH. The evaluation of these RPA template sets in various murine models demonstrated their suitability for the analysis of the above chemokines both under constitutive and infection-induced conditions. To reduce the personal radiation hazard, we found that 32P could be replaced by 33P without any loss of assay-sensitivity. These new RPA multi-probe sets provide valuable tools for the simultaneous quantitative determination of gene expression of multiple murine chemokines of both constitutive and inducible type.

Chemokines, natural killer cells and granulocytes in the early course of Leishmania major infection in mice.

In the present study the early recruitment of leukocytes into the infected skin and into the draining lymph node (LN) was investigated after subcutaneous infection of mice with Leishmania major promastigotes. Flow cytometric analysis of cells recovered from the infected skin revealed that GR-1+ granulocytes were present as early as 10 h after infection, thus representing the first leukocyte population to be recruited to the site of infection. The migration of granulocytes was shown to be associated with a rapid mRNA expression of the neutrophil-attracting chemokine KC in the infected skin. Moreover, L. major promastigotes were found to produce factor(s) that are chemotactic for human neutrophils in vitro. Experiments with human neutrophils revealed that these cells are able to phagocytose the parasites. Natural killer (NK) cells appeared at the site of infection 24 h after infection. The migration of NK cells in resistant mice was found to correlate with the expression of the NK cell-activating chemokine IP-10. Treatment of susceptible BALB/c mice with recombinant mouse IP-10 resulted in a significantly increased NK cell cytotoxic activity in the draining LN. These data suggest that both the early chemokine gene expression and the production of chemotactic factors by the parasites themselves regulate the site-directed migration and activation of cells of the innate immune response, and suggest a role of chemotactic factors in the early defense against the parasites.

Early gene expression of NK cell-activating chemokines in mice resistant to Leishmania major.

Susceptibility of mice to Leishmania major is associated with an insufficient NK cell-mediated innate immune response. We analyzed the expression of NK cell-activating chemokines in vivo during the first days of infection in resistant and susceptible mice. The mRNA expression of gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1 day after infection in the draining lymph nodes of resistant C57BL/6 mice but not in those of susceptible BALB/c mice. In vivo local treatment of BALB/c mice with recombinant IP-10 shortly after infection resulted in an enhanced NK cell activity in the draining lymph node. The data suggest that although the recruitment of NK cells is normal in susceptible mice, the lack of NK cell-activating chemokines is a factor resulting in a suboptimal NK cell-mediated defense.

Type 1 interferon (IFNalpha/beta) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite.

Type 2 nitric oxide synthase (NOS2) is required for the Th1-dependent healing of infections with intracellular microbes, including Leishmania major. Here, we demonstrate the expression and define the function of NOS2 during the innate response to L. major. At day 1 of infection, genetic deletion or functional inactivation of NOS2 abolished the IFNgamma and natural killer cell response, increased the expression of TGFbeta, and caused parasite spreading from the skin and lymph node to the spleen, liver, bone marrow, and lung. Induction of NOS2 was dependent on IFNalpha/beta. Neutralization of IFNalpha/beta mimicked the phenotype of NOS2-/- mice. Thus, IFNalpha/beta and NOS2 are critical regulators of the innate response to L. major.

In vivo blocking of L-selectin rescues BALB/c mice from fatal Leishmania major infection.

Susceptibility and resistance to experimental Leishmania major (L. major) infection in mice are associated with a Th2- or Th1-type response, respectively. We have previously shown that immunological events occurring within the first 24 h after infection in the lymph node (LN) draining the site of parasite challenge are critical for the development of either type of T-cell responses. In the present study we manipulated these events by preventing the entry of naive lymphocytes into the draining LN by injecting BALB/c mice with a single dose of the anti-L-selectin mAb MEL-14 one day prior to infection with L. major. In contrast to control BALB/c mice, in MEL-14 treated animals the primary lesion healed 12 weeks after infection. The parasite load in the spleen and lymph nodes of MEL-14 treated mice was significantly reduced. The healing was found to be associated with an increased production of IFN-gamma and with a decrease in IL-4 production by LN cells. We observed a dramatic decrease in cellularity in the draining LN in Mel-14 treated L. major-infected mice within the first week of infection. Moreover, the cells in the LN of MEL-14 treated mice were highly enriched in activated cells as well as in cell influx in the draining LN after local L. major infection of BALB/c mice prevents fatal disease. The data suggest the MEL-14-induced enrichment of the draining LN in memory and activated cells is fundamental for the initiation of a protective Th1-type response.

Improved technique for cannulation of the murine thoracic duct: a valuable tool for the dissection of immune responses.

Several experimental approaches have been used in the past for the cannulation of the thoracic duct in mice. Most, however, are characterized by a modest reproducibility and a low lymph yield. Here, we describe a cannulation technique modified with respect to the anesthesia, the use of a silicone cannula and a simple and efficient intraabdominal fixation of the drain. Surgery averaged 45 min with an intra- and postoperative mortality rate of zero. Postoperatively, mice were given access to an exercise wheel allowing increased mobility and consequently a good lymph flow, thus maintaining the function of the cannula. The mice yielded a mean of 29.3 ml/24 h (range 8-40 ml) thoracic duct lymph, which contained a mean of 2.2 x 10(6) lymphocytes/ml during the first 24 h, decreasing to 0.1 x 10(6) lymphocytes/ml on the 2nd day after cannulation. Patency of the cannulae was 100% after 3 days. Interestingly, we have detected strain dependent differences in the anatomy of the thoracic duct in the mouse and these need to be considered when cannulation procedures are attempted.

Control of Leishmania major infection in BALB/c mice by inhibition of early lymphocyte entry into peripheral lymph nodes.

A single i.p. injection with the anti-CD62L (anti-L-selectin) mAb Mel-14 before parasite challenge protected BALB/c mice from the otherwise lethal infection with Leishmania major. The Mel-14 mAb treatment resulted in a significant (>90%) decrease in cellularity of the popliteal lymph node (PLN) with a decrease in the proportion of CD4+ cells and an increase of the proportion of B220+ cells. Furthermore, both activated cells (CD25+ and CD69+) and cells of the memory phenotype (CD45RBdull CD44high) were significantly enriched in PLN from Mel-14-treated BALB/c mice. After infection with L. major, the otherwise massive cellular infiltration in the draining PLN was completely blocked in the Mel-14-treated mice, and in these animals the high representation of both activated and memory cells in PLN remained characteristic for the first days of infection. The protective effect was found to be associated with a markedly increased production of IFN-gamma and with a decrease in IL-4 production upon restimulation of PLN and spleen cells with L. major Ag in vitro. The cured mice were found to be resistant against a secondary challenge with the parasites. These data suggest that the induction of a nonprotective Th2 response to L. major is associated with the entry of lymphocytes from the recirculating pool into the draining LN. The Mel-14-induced changes in the lymphoid microenvironment of the draining peripheral LN appear to favor the development of a protective Th1 cell-mediated immune response against the parasite.

Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis.

The abundance of macrophages in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL) lesions and differences in the composition of T cell subsets indicate involvement of cell-specific chemotaxis processes. The expression of macrophage chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and -1 beta, RANTES (regulated on activation, normal T cell expressed and secreted), I-309, and interleukin-8 were investigated in lesions of patients with LCL or DCL. In LCL, high levels of MCP-1 and moderate levels of MIP-1 alpha were detected. In DCL, MCP-1 expression was significantly lower and MIP-1 alpha expression was predominant. All other chemokines investigated were minimally expressed or absent. These findings suggest that MCP-1 and MIP-alpha are responsible for the recruitment of macrophages and T cells in cutaneous leishmaniasis. The results show that self-healing LCL is associated with higher levels of MCP-1, which may stimulate macrophage microbicidal mechanisms, and nonhealing DCL is associated with higher levels of MIP-alpha.

Leishmania major infection: the overture.

Local infection of mice with Leishmania major results in either healing or death depending on the preferential action of Th1 or Th2 T helper cells, respectively. Although the parasite-induced T-cell responses and their consequences for the disease are well understood, relatively little is known about the initial events that kindle the adaptive immune response. Werner Salbach and Tamás Laskay here discuss how differences in parasites spreading from the site of infection to different immune organs during the first 10-24 hours and, in consequence, the 'where and when' of the first encounter of Leishmania with the cells of the immune system may well be the starting point for the development of resistance or susceptibility.

Early parasite containment is decisive for resistance to Leishmania major infection.

We investigated the early spread of Leishmania major in various mouse strains. In BALB/c mice, which are extremely vulnerable to L. major infection, the parasites disseminated within 10-24 h from the site of subcutaneous footpad infection in to the popliteal lymph node, spleen, lung, liver and bone marrow. Application of recombinant (r)IL-12 prior to infection prevented the early dissemination of parasites into visceral organs and the animals healed the infection. In three mouse strains tested, C57BL/6, CBA/J and C3H/HeJ, which are all resistant to L. major infection, the parasites remained localized in the footpad and in the draining LN for 3 days without evidence of dissemination. In C57BL/6 mice, depletion of NK1.1+ cells or neutralization of interferon (IFN)-gamma prior to infection led to rapid parasite spreading with kinetics similar to those seen in susceptible animals. Depletion of either CD4+ or CD8+ T cells in vivo prior to infection did not alter the kinetics of dissemination in any mouse strain tested. Experiments with severe-combined immunodeficient mice provided further evidence that parasite containment depends on natural killer cells and IFN-gamma, but is independent of T cells. The finding that all resistant mouse strains restrict the spread of the parasites within the first 24 h after infection strongly suggests that early parasite containment is closely associated with a resistant phenotype. The data show that local restriction of parasites in the pre-T cell phase of the infection is mediated by the innate immune system and suggest that this function plays an important role in the development of a protective T cell response.

Detection of cutaneous Leishmania infection in paraffin-embedded skin biopsies using the polymerase chain reaction.

In this study the polymerase chain reaction (PCR) with previously developed oligonucleotide primers was used to detect Leishmania aethiopica in paraffin-embedded skin biopsy specimens. The Leishmania-specific 120 base pair fragment of the kinetoplast deoxyribonucleic acid (kDNA) minicircles has been amplified from all parasitologically or histologically confirmed cases of cutaneous leishmaniasis (CL), as demonstrated by gel electrophoresis and hybridization with L. aethiopica kDNA. Control specimens from patients with skin diseases other than CL were all negative. Using PCR, Leishmania were demonstrated in the skin lesions of 7 cases in a group of 40 patients in whom the parasites could not be demonstrated by histopathology or culture in vitro although lesions were clinically suggestive of CL. These data indicate that PCR, carried out on DNA extracted from formalin-fixed and paraffin-embedded tissue specimens, is a valuable method for the diagnosis of CL, especially in chronic cases where the parasite load in the lesion is low.

Natural killer cells participate in the early defense against Leishmania major infection in mice.

In this study the role of natural killer (NK) cells in the course of experimental Leishmania major infection was investigated. NK cells in genetically resistant C57BL/6 mice were depleted by in vivo administration of anti-asialo-GM1 or anti-NK1.1 antibodies. A marked exacerbation of the infection was found in the NK-depleted mice within the first two weeks of infection. Both the local tissue swelling and the number of parasites in the lesions were significantly higher than in normal animals. Lymph node cells taken from infected NK-depleted mice released less interferon-gamma (IFN-gamma) when cultured in vitro. As an alternate approach we have used poly I:C treatment in order to activate NK cell activity in vivo in BALB/c mice, which are genetically susceptible to L. major infection. Poly I:C treatment led to milder symptoms and to a significantly lower parasite burden in the early course of infection. Lymph node cells from infected and poly I:C-treated BALB/c mice released higher amount of IFN-gamma in vitro than cells from control mice. These data show that NK cells are active participants in the non-specific phase of anti-leishmanial activity in the control of parasite multiplication early in the course of L. major infection in mice.