Martin E. A. Mielke

Cytokines in the induction and expression of T-cell-mediated granuloma formation and protection in the murine model of listeriosis

Summary: Lymphocyte‐mediated inflammation is a hallmark of autoimmune diseases, such as multiple sclerosis, Crohns disease, rheumatoid arthritis and sarcoidosis. However, this type of inflammation probably developed under evolutionary pressure from pathogenic microorganisms, such as mycobacteria and other intracellular infective agents. One such pathogen, the gram‐positive bacterium Listeria monocytogenes (L. monocytogenes), induces a cascade of tissue alterations that ultimately results in the eradication of the bacteria associated with a granulomatous response. Consequently, murine listeriosis has been established as a model to analyze not only T‐cell‐dependent antibacterial protection but also T‐cell‐mediated mononuclear inflammation in parenchymal organs. Extensive studies of the molecular basis of the latter phenomenon led to the conclusion that the most decisive step from non‐specific microabscess formation to granulomatous inflammation is the activation of non‐specifically invading CD4+ T cells, which results in high local concentrations of TNF‐α and IFN‐γ in the presence of IL‐2. This in turn induces CD11b‐independent mechanisms of intraparenchymal monocyte accumulation. Because any attempt to neutralize the effects of TNF‐α and IFN‐γ to modulate T‐cell‐mediated inflammation will also dramatically decrease host resistance, other anti‐inflammatory strategies based on the modulation of TNF‐α and IFN‐γ‐induced mechanisms of monocyte accumulation must be developed. Recalling the classical work by Dienes & Schoenheit on the induction d bacterial allergies (1), the cytokine phenotype of granuloma formation also has implications as regards the most potent adjuvant environment for the development of a T‐cell response. The murine listeriosis model is the basis for all conclusions in this article on the role of cytokines in the induction and expression of T‐cell‐mediated inflammation and. as we will show, promises to yield still more insights into the rational design of vaccines.

The Role of Cytokines in Experimental Listeriosis

Listeria monocytogenes is a Gram-positive, intracytoplasmatically replicating pathogen that elicits host reactions which are very similar in man and rodents. Using murine listeriosis as a highly reproducible and convenient experimental model for studying the immune response to infections with facultative intracellular bacteria, Mackaness developed the concept of T cell-mediated macrophage activation as the pivotal mechanism in host defense against this type of infectious agents. Continued research in listeriosis itself, however, provided paradoxical findings that challenged the original dogma. In particular, the finding that T cell-mediated inflammatory events, like DTH and granuloma formation, can be dissociated from protective effector mechanisms has provided a new impetus and experimental access to characterizing the molecular mediators responsible for these diverging phenomena. This review first summarizes the cellular basis for the dichotomy of immunological phenomena outlined above and will then relate recent findings on cytokine expression in infected tissues to these dual categories of the host response to infection. The authors will focus on data obtained from in vivo experiments and draw on evidence from in vitro systems only when appropriate in vivo verification is still lacking. The data presented will cover the developments made in the field of cytokine research since our previous review in 1981 (Rev. Infect. Dis. 3: 1221-1250). Detectable numbers of listeria-specific T cells become apparent on day 4 to 5 of a primary infection. Whereas the localized and sustained release of TNF and IFN-gamma mediated by CD4+ cells seems to be the focusing event triggering mononuclear cell accumulation, the coincidental eradication of bacteria critically depends on CD8+ and/or CD4-CD8-Thy1+ cells. Their effector functions, however, remain obscure, since cytokines cannot be identified that will substitute for their presence. None of the cytokines studied thus far has been demonstrated to effectively cure an established infection. In addition, the increased production of cytokines characteristic of an anamnestic response (IL-2, IL-3, IL-4, IFN-gamma and TNF) can be dramatically reduced by depleting CD4+ T cells without any effect on the animals ability to eradicate high lethal doses of bacteria and Listeria-specific CD8+ T cells can mediate protection even in the presence of neutralizing antibodies to IFN-gamma. In conclusion, the murine model of Listeria infection provides an interesting experimental approach for the development of immunotherapeutic strategies aimed at reducing T cell-mediated immunopathology without interfering with innate resistance and T cell-mediated cure and prevention of disease.

Progress in TB research: Robert Koch's dilemma revisited

Robert Kocl~, when describing the causative agent of tuberculosis (TB) in 1882, was first to realize that the bacillus is not all there is to tuberculous disease. When he went on to introduce therapeutic immuaomodulation using a glycerol-extract of M. tuberculosis, he was successful in treating tuberculous skin disease; however, in advanced cases of pulmonary disease, his cure caused massive tissue destruction and treatment failureL Kochs dilemma the fine line between protection and pathology in TB still exists a century after his discoveries. The occurrence of multi-drug resistant (MDR) TB strains, and the lack of efficient immunization procedures December 1993 marked ibe I50ih anniversary o f tke birth o f Robert Koch. To commemorate his milestone contributions to immunolo~, and microbiology a symposi:,;m* was be!d recently to discuss the interface between the immune system and the ~:,er tenacious Mycobacterium tuberculosis.

Tailoring host immune responses to Listeria by manipulation of virulence genes: the interface between innate and acquired immunity

Although attenuated strains of microbial pathogens have triggered vaccine development from its origin, the role of virulence factors in determining host immunity has remained largely unexplored. Using the murine listeriosis model, we investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes. We intentionally deleted specific genes of Listeria monocytogenes, including those encoding the positive regulatory factor (prfA), hemolysin (hly), the actin nucleator (actA), and phospholipase B (plcB). The resulting strains showed decisive differences in their immunogenic properties. In particular, we identified a double-deletion mutant that retained Listerias profound ability to induce protective CD8(+) T cells, but that is strongly attenuated and exhibits a significantly reduced ability to induce CD4(+) T cell-mediated inflammation. We conclude that this mutant, L. monocytogenes DeltaactADeltaplcB, is at present the most promising mutant for a bacterial vaccine vector and is able to safely induce potent CD8(+) T cell-mediated immunity.

The role of T cell subpopulations in cell mediated immunity to facultative intracellular bacteria

SummaryThis brief review summarizes the experimental data which underly the classic concept of anti-bacterial cell mediated immunity and will integrate recent developments focusing on results obtained byin vivo studies in the model of rodent listeriosis.ZusammenfassungDiese kurze Übersicht faßt die experimentellen Daten zusammen, die dem klassischen Konzept antibakterieller zellvermittelter Immunität zugrunde liegen, und schließt die neuesten Erkenntnisse unter besonderer Berücksichtigung vonIn-vivo-Untersuchungen am Modell der Listeriose von Maus und Ratte ein.

Primary isolation of Mycobacterium tuberculosis on blood agar during the diagnostic process for cat scratch disease

We here present a case of necrotizing lymphadenit is from which we isolated Mycobacreriunr tuberculosis on blood agar within 13 days during the diagnostic process for cat scratch disease. A 82-year-old woman was admitted with a 4-month history of enlarged right supraclavicular lymph nodes. Ultrasonography revealed three enlarged, partially suppurated nodes. An incision biopsy was performed and yielded pus which remained sterile in rout ine microbiological workup. Cultures for mycobacteria were not performed at this time. Histological examinat ion revealed a granulomatous necrotizing inf lammation with giant cells. Acid-fast bacilli were not detected, and the findings were found suggestive of either tuberculosis or cat scratch disease. Chest roentgenogram and computed tomogram of the thorax did not reveal any signs of tuberculosis, but thickening of both apices and a large left pleural effusion. The intracutaneous tuberculin skin test was positiv at t_ TU. After 4 weeks, a fistula had developed at the incision area so that a second operat ion was performed and the node extirpated. Biopsy specimens were sent to our laboratory to be examined for Bartonetla henselae and to a second laboratory for the detection of mycobacteria. We received the lymph node biopsy material in saline a few hours after extirpation. The specimen was minced and homogenised in 2 ml saline, 0.2 ml ahquots were plated onto solid media. including trypticase soy agar (BBL Microbiology Systems, Cockevsville, MD, USA), supplemented with 5% sheep erythrocytes tb lood agar), chocolate and Columbia agar (Unipath LTD, Basingstone. UK), both supplemented with 5/0 human erythrocytcs, and incubated at 37~ in a 7% CO 2 atmosphere for up to 6 weeks. One aliquot was subjected to PCR for B. henselae [1] with negative results. After 13 days, approximately 50 small, non-pigmented, rough colonies grew on blood agar. At this time, growth was not observed on Columbia and chocolate agars; however, after further 12-14 days of incubation, several minute colonies grew also on the latter media. The bacteria were not properly labelled bv Gramstain, while Ziehl-Neelsen stain revealed slender, granulated, acidfast bacilli. The isolate was identified as M. tuberculosis bv means of RNA-amplification (AMPLIFIED, Gen-Probe. San Diego, CA. USA), DNA-RNA-hybridisation (ACCU-Probe, Gen-Probe. San Diego. CA, USA), and biochemical tests. Subcultures grew within 10 days with a typical colony morphology on solid Middlebrook and Loewenstein-Jensen media: the isolate was susceptible to all antituberculous drugs tested. Upon further subcultivation on blood agar, the isolate grew even more rapidly, i.e., within 6--7 days. Recently, a rapid growing M. tuberculosis strain has been described in the USA [2], and we tested whether our isolate might belong to this group of mycobacteria, However, when inoculated in comparable numbers, the M. tttberculosis reference strata H37 Rv [3], which serves as a control strain in susceptibility testing [4] and has been serially passaged on Loewenstein-,lensen agar for over 20 )ears in our laboratory, also grew on blood agar within a similiar period of time. Hence, the patients isolate did not possess any special properties with respect to its nutritional requirements or growth kinetics. Our report documents the isolation of M. tuberctdosts from a clinical specimen under conditions used for the culture of B. hense&e [5 7]. As both M. tuherculosis and B. henselae are common agents of chronic lymphadenitis, microbiologists should be aware of the possibility to culture M. tuberculosis during the diagnostic process for cat scratch disease. M, Arvand, M. E. A. Mielke. T. Weinke, 7~ Regnath, H. Hahn

Role of the systemic cellular immune response in the pathogenesis of Helicobacter pylori-associated duodenal ulcer

20 patients colonized by H. pylori (9 with duodenal ulcer, 11 with antral gastritis) were examined for the presence of a systemic cellular immune response to H. pylori using an in vitro lymphocyte transformation assay. Infiltration of the antral mucosa by myelomonocytic cells was assessed by immunohistology as a parameter of local cellular immune response. A systemic H. pylori-induced cellular reaction could be demonstrated in 11 patients all of whom showed an intense myelomonocytic infiltration of the antral mucosa. In this group 7 of 9 duodenal ulcers occurred. The findings support the hypothesis, that a specific cellular immune response might be involved in the pathogenesis of H. pylori-associated gastritis and duodenal ulcer. A protective role of systemic cellular immunity to H. pylori seems rather unlikely.

Protective immunity and granulomatous inflammation is mediated in vivo by T cells reactive to epitopes common to avirulent and listeriolysin-negative mutants of Listeria monocytogenes

The ability of several listeriolysin O-negative mutants of the EGD and NCTC 7973 strains of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. T cell lines from different inbred mouse strains and derived T cell clones elicited by L. monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing revealed no differences between listeriolysin-positive and -negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined CD4+ T cell-mediated granuloma formation in the livers of mice previously immunized with viable bacteria of the virulent strain. Granulomatous inflammation could be elicited by iv application of heat-killed bacteria of listeriolysin-positive and of -negative bacteria. Protective immunity to listerial infections and granulomatous inflammation therefore appears to be mediated by T cells recognizing epitopes on listerial antigens that are shared by both pathogenic and nonpathogenic Listeria strains.

The mRNA-Phenotype of Granuloma Formation: CD4+ T Cell-Associated Cytokine Gene Expression during Primary Murine Listeriosis

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.

Listeriolysin Negative Mutants of Listeria monocytogenes Specifically Stimulate T-Lymphocytes Mediating Protection and Granulomatous Inflammation

The ability of several listeriolysin O negative mutants of the virulent EGD strain of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. A T cell line and a derived clone specific for Listeria monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing showed no difference between listeriolysin positive and negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined T cell mediated granuloma formation in the livers of mice previously immunized with viable bacteria. Granulomatous inflammation could be elicited by i.v. application of heat killed bacteria of listeriolysin positive as well as of negative bacteria. Thus, the expression of the heat induced 60KD listeriolysin protein is not a prerequisite for the stimulation of Listeria-specific T lymphocytes mediating protection and granuloma formation.