- Email: [email protected]
S.H.E. Kaufmann:
67th FORUM Reply from North
and Medina
“Yes. I believe that the physiological processes associated with superior genetic resistance in the lung are highly complex and almost certainly under the control of multiple genes. One need only look at the histological complexity of individual lung lesions to obtain an appreciation of the complexity of the host-pathogen interaction in this organ. The results show that resistant and susceptible mice are equally capable of stabilizing infection in the liver and spleen (organs in which Nramp1 is expressed), but not in the lungs after the onset of expression of specific immunity. Therefore, immunity is less efficiently expressed only in the lungs of susceptible mice. This could result, on the one hand, from the extravasation of fewer CD4 T cells (Thl) into lesions because of the production of smaller quantities of appropriate proinflammatory cytokines. Or, it could be caused by a defect in macrophages that makes them less capable of processing and presenting Ag and consequently of being microbicidally activated by adequate numbers of Thl cells. There are numerous other possibilities. As for how we might go about identifying the resistant gene(s), we would need to first map it to a location on a given chromosome(s) according to its proximity to known marker genes in congenitally resistant lines of mice that we would produce by appropriate backcrossing. Unfortunately, even if only one gene were involved, this would take 3-4 years, given that we would need to select progeny that survive M. tuberculosis infection for more than 100 days from 12 consecutive backcrosses. We currently are looking at recombinant inbred strains with the hope of shortening the process.” Commentary
by D. Miinnel:
IN IMMUNOLOGY p75TNFR participation in bacterial clearance is supported by the finding of increased mortality over 6 days in p75TNFR-deficient mice compared to wild type mice by Erickson et al. (Nature, 372, 560, 1994).
Reply from Unanue “I agree that the effects of Note that we could not reverse anti-TNF treatment with IFNy. expression was restored, but infection persisted.”
TNF are diverse. all the effects of Indeed, class II susceptibility to
Commentary
Kaufmann:
by J. HessL3.H.E.
On Unanue Your interesting review offers deep insight ir,to the immediate early stage of listeriosis, involving not only active bacterial killing, but also apoptosis of target cells. The conclusions about apoptosis in control of listerioris are based on the investigations by Rogers et al. who employed the TUNEL assay. Recently, evidence has been presented that TUNELpositive nuclei of hepatocytes are also observed during necrosis and after non-specific DNA fragmentation in insufficiently fixed autolytic livers (Grasl-Kraupp et al., Hepatoiogy, 21, 1465-1468, 1995). We therefore, wonder whether you have additional experimental evidence for apoptosis in Listeria-infected livers, such as agarose gel electrophoresis or electron microscopy (Bohlinger et al., Am. J. Pathol., 149, 1381-1393, 1996) to gain further insights into the type of cell death of Listeriainfected hepatocytes in vivo.
Reply from Unanue
On Unanue E. Unanue’s work clearly shows that TNF and IL12 release by macrophages and the resulting IFNy production by T or NK cells stand out as the protective mechanisms early in the bacterial infection of macrophages. How exactly TNF participates in this interplay, however, is still unclear. Because in such cell-cell interactions of activated macrophages with activated T/NK cells all participating cell types are TNF producers, requirement of the p75TNF receptor (TNFRII) triggering could be possible, maybe preferentially by membrane TNF. This possibility could be tested by studying the combined effect of IFNy and membrane TNF on Listeria survival in macrophages or by using macrophages of p75TNFR-deficient mice. The idea of
“The study by Rogers included the analysis of DNA fragmentation. Ultrastructural studies also substantiated the presence of apoptotic nuclei (unpublished).”
On Kurlander
and Nataraj
This highly interesting paper provides an explanation for preferential processing through MHC Ia or MHC Ib by viable vs. killed L. monocytogenes, respectively. Consistent with this, one of us (S.H.E.K.) had observed that generation of MHC Iarestricted CD8 T cells was facilitated by using viable L. monocytogenes for restimulation and assay (Kaufmann et al., J. Exp. Med., 164, 363-368, 1986). We
IMMUNITY
TO INTRACELLULAR
wonder whether you have any evidence as to whether MHC Ib processing can also originate from the cytosolic compartment or whether it is restricted to the endosomal compartment. In other words, whether MHC Ib is loaded with peptides from viable cytosolic L. monocytogenes. And if so, do you know whether this is a TAP-dependent process? We wonder whether, in addition to the MHC Ibrestricted CD8 T cells, conventional MHC class-I restricted CD8 T cells could also be activated by antigens originating from the endosome, because alternative pathways have been described from the endosome to the conventional MHC class I processing pathway. Such an assumption would be consistent with earlier findings.
Reply from Kurlander “We clearly must acknowledge the seminal early contribution of Dr. Kaufmann and his collaborators in recognizing ‘MHC-unrestricted’ CTL recognition of L. monocytogenes. It is still unclear how HAA/lemA is processed and presented. New H2M3VVZ biosynthesis is clearly required, and both we (unpublished data) and others (Lenz et al., 19961 have noted that presentation is greatly diminished but not absolutely blocked in Tap2-deficient animals. Simple shunting of antigen from endosome to cytoplasm clearly is not sufficient for initiation of processing and presentation; hence, we believe processing proceeds either by late shunting (i.e. after several hours) of endosomally preprocessed antigen into the cytoplasm for endogenous processing or by a novel pathway of antigenic peptide binding to H2M3ti within the endosome. Additional studies will be required to distinguish between these possibilities.”
On White et al. The article by White et al. reviews the importance of CD8 T cells in murine infection by L. monocytogenes/S. typhimuriun&f. bovis BCG or M. tubercuiosis. The in vivo relevance of CD8 T cells is still questionable in murine S. typhimurium infection. Nevertheless, we have evidence that attenuated S. typhimurium equipped with phagolysosomal escape function did not alter the course of infection in &mdeficient mice in comparison with the control strain (Hess et al., J. Immunol., 156, 3321-3326, 1996). These data suggest that, even in this case, CD8 T cells were not required for Salmonella clearance. As a corollary, we think that clearance of primary S. typhimurium infection only requires a CD4 T-cellmediated immune response. In secondary Sulmonella
BACTERIA
609
ssp. infection, the number of stimulated CD8 T cells increases (Pope et al., Immunology, 81, 177-182, 1994) but the relevance to host defence still has to be clarified.
Reply from White et al. “The experimental results of Hess and Kaufmann demonstrating that phagosomal escape did not enhance Salmonella virulence in pZrn knockouts are consistent with the interpretation that clearance of primary Salmonella infection is independent of CD8+ T cells (Hess et al., J. Immuno/., 156, 3321-3326, 1996). However, these recombinant Salmonella lack the actin-polymerizing activity and intracellular spread capability that are essential virulence determinants of Listeria and Shigella infection. Thus, cytoplasmic Salmonella are still trapped within the initially infected cell. The infected cells will eventually die in a fashion that is independent of the activities of CD8’ T cells, although CD8+ T-cell killing could result in destruction of the infected cell prior to extensive replication of Salmonella.”
On North and Medina We consider it highly interesting that resistance/susceptibility to lung tuberculosis does not follow that to BCG in different mouse strains. Do you know whether genes have been identified in regions near the Nramp gene which could cosegregate in BALB/c versus DBA mice? Such an impact of cosegregating genes could be avoided by using Nramp gene knockout mice which have been generated by Vidal et al. It is apparent to us that the data presented clearly show that the difference is related to the lung and knowledge about potential candidate genes which might interfere with Nramp in control of lung tuberculosis would be an interesting line of future investigation.
Reply from North and Medina “We agree that it would be interesting to look at resistant mice in which Nrampl has been functionally deleted by homologous recombination. However, the results of our study of 100 F progeny of BALB/c (Nramp1 sus) and DBAII (NramplfeSl mice show that Nramp7 segregates independently of resistance to M. tuberculosis, in that heterozygous F mice, as well as F, mice homozygous for each Nramp1 allele, died randomly between 80 and 400 days after receiving IO5 M. tuberculosis i.v. This would argue against
610
67th FORUM
linkage to Nramp7, ready for surprises.”
although
we are always
IN IMMUNOLOGY On Brombacher
and Kopf
Since performing the research discussed in this article, we have become aware of an earlier paper by Musa et al. (Infect. Immun., 55, 1863-1866, 1987) showing that mice now known to be homozygous for the NramplreS allele are not more resistant to h4. tuberculosis infection than mice homozygous for the NramplSUS allele, according to bacterial growth in the lung, liver and spleen over 50 days of infection. Although congenic mice were not used in the study, the results leave little doubt that the role of Nrampl in anti-tuberculosis resistance is questionable. We apologize for having missed this paper. It is apparent that it has escaped the notice of most people active in the field.
I enjoyed reading your paper, particularly the issue that either IL10 or IL4 may act as counterregulators of IFNy-mediated response, depending on the pathogen and the resulting host pathology and immunity. Similarly, in Candida albicans murine infections, we have seen that either IL4 or IL10 may oppose the lL12-dependent Thl response, depending on the yeast/host combination and the ensuing immunopathology. In the case of Listeria, ILlO, more than lL4, appears to act as a counterregulatory cytokine in infection, as the data on KO mice suggest. However, I was wondering whether IL4 and/or IL10 could be produced in mice highly susceptible to infection, such as in STAT-l-‘or IFNy-‘or IFNyR-‘mice. Is the susceptibility of these mutant mice reversed by IL4 or IL10 neutralization? And, in either case, could the result be related to the type of infection ?
Commentary
Reply from Brombacher
General commentary
by R. North and E. Medina:
by L. Romani :
On Hess and Kaujinann I find your results on Salmonella enterica infection very important, particularly with respect to the differences between the different intracellular pathogens. One most impressive finding appears to be the very efficient bacterial control observed in the first 20 days of infection that was clearly IFNydependent and T-cell-independent. However, TCRP-‘- mice efficiently control the infection at 20 days postinfection, at a time when IFNy-deficient mice succumb to infection. It appears that other alternative cellular sources of IFNy are at work or additional mechanisms controlling infection are operative. Would you like to comment on the early phase control of infection?
Reply from Hess and Kaufmann “We think that in the early phase (until day 20 p.i.) NK ceils are the dominant contributors to the control of Salmonella enterica infection. Consistent with this notion is the course of salmonellosis in Ap-Iand IFNyR-‘--deficient mutants. After day 20 p.i., the mutant mice suffer from severe chronic infection or die of salmonellosis, respectively. In addition, RAG-l-/animals which are devoid of functional T and B lymphocytes have a longer survival time than IFNyR-I- mice infected with S. enterica. These data suggest that NK cells may control Salmonella infection via IFNy secretion and/or NK activity.”
“We have no data on STAT-l-/or IFNy-Imice, but we recently et-formed Listeria infection studies in IFNyR- P- IL4-/- double-deficient mice. Two experiments (5 mice/group) with low dose infection (around 200 CFU) were performed by us. In the first experiment, all double-deficient mice died on the same day as IFNyR deficient mice. In the second experiment, most double-deficient mice died with a delay of one day, but one survived. We now control whether the surviving mouse has immunity to a secondary response. However, in a recently published study by S. Kaufmann’s group using anti-ILCtreated IFN$V/- mice, all mice survived and cleared bacteria after a similar low infection dose. At the moment, we cannot explain why anti-114 treatment has stronger effects, but, taken together, these data indicate that depletion of endogenous IL4 has at least some protective influence in IFNyR-‘- mice. The cellular source for the increased resistance has to be identified and may have influenced innate and specific responses. The second part of your question, does IL10 neutralization reverse the phenotype in IFNyR-I- mice, is also very interestin We are in the process of establishing ILIO- 3.- IFNyR-I- double-deficient mice and we hope to answer this question. Nevertheless, as extracted from this Forum showing that infection with a certain pathogen may activate or suppress particular immunoregulatory cell types (and it seems we are finding more and more of them), I do not doubt that the answers we are getting are indeed related to the type of infection.”
and Medina
“Yes. I believe that the physiological processes associated with superior genetic resistance in the lung are highly complex and almost certainly under the control of multiple genes. One need only look at the histological complexity of individual lung lesions to obtain an appreciation of the complexity of the host-pathogen interaction in this organ. The results show that resistant and susceptible mice are equally capable of stabilizing infection in the liver and spleen (organs in which Nramp1 is expressed), but not in the lungs after the onset of expression of specific immunity. Therefore, immunity is less efficiently expressed only in the lungs of susceptible mice. This could result, on the one hand, from the extravasation of fewer CD4 T cells (Thl) into lesions because of the production of smaller quantities of appropriate proinflammatory cytokines. Or, it could be caused by a defect in macrophages that makes them less capable of processing and presenting Ag and consequently of being microbicidally activated by adequate numbers of Thl cells. There are numerous other possibilities. As for how we might go about identifying the resistant gene(s), we would need to first map it to a location on a given chromosome(s) according to its proximity to known marker genes in congenitally resistant lines of mice that we would produce by appropriate backcrossing. Unfortunately, even if only one gene were involved, this would take 3-4 years, given that we would need to select progeny that survive M. tuberculosis infection for more than 100 days from 12 consecutive backcrosses. We currently are looking at recombinant inbred strains with the hope of shortening the process.” Commentary
by D. Miinnel:
IN IMMUNOLOGY p75TNFR participation in bacterial clearance is supported by the finding of increased mortality over 6 days in p75TNFR-deficient mice compared to wild type mice by Erickson et al. (Nature, 372, 560, 1994).
Reply from Unanue “I agree that the effects of Note that we could not reverse anti-TNF treatment with IFNy. expression was restored, but infection persisted.”
TNF are diverse. all the effects of Indeed, class II susceptibility to
Commentary
Kaufmann:
by J. HessL3.H.E.
On Unanue Your interesting review offers deep insight ir,to the immediate early stage of listeriosis, involving not only active bacterial killing, but also apoptosis of target cells. The conclusions about apoptosis in control of listerioris are based on the investigations by Rogers et al. who employed the TUNEL assay. Recently, evidence has been presented that TUNELpositive nuclei of hepatocytes are also observed during necrosis and after non-specific DNA fragmentation in insufficiently fixed autolytic livers (Grasl-Kraupp et al., Hepatoiogy, 21, 1465-1468, 1995). We therefore, wonder whether you have additional experimental evidence for apoptosis in Listeria-infected livers, such as agarose gel electrophoresis or electron microscopy (Bohlinger et al., Am. J. Pathol., 149, 1381-1393, 1996) to gain further insights into the type of cell death of Listeriainfected hepatocytes in vivo.
Reply from Unanue
On Unanue E. Unanue’s work clearly shows that TNF and IL12 release by macrophages and the resulting IFNy production by T or NK cells stand out as the protective mechanisms early in the bacterial infection of macrophages. How exactly TNF participates in this interplay, however, is still unclear. Because in such cell-cell interactions of activated macrophages with activated T/NK cells all participating cell types are TNF producers, requirement of the p75TNF receptor (TNFRII) triggering could be possible, maybe preferentially by membrane TNF. This possibility could be tested by studying the combined effect of IFNy and membrane TNF on Listeria survival in macrophages or by using macrophages of p75TNFR-deficient mice. The idea of
“The study by Rogers included the analysis of DNA fragmentation. Ultrastructural studies also substantiated the presence of apoptotic nuclei (unpublished).”
On Kurlander
and Nataraj
This highly interesting paper provides an explanation for preferential processing through MHC Ia or MHC Ib by viable vs. killed L. monocytogenes, respectively. Consistent with this, one of us (S.H.E.K.) had observed that generation of MHC Iarestricted CD8 T cells was facilitated by using viable L. monocytogenes for restimulation and assay (Kaufmann et al., J. Exp. Med., 164, 363-368, 1986). We
IMMUNITY
TO INTRACELLULAR
wonder whether you have any evidence as to whether MHC Ib processing can also originate from the cytosolic compartment or whether it is restricted to the endosomal compartment. In other words, whether MHC Ib is loaded with peptides from viable cytosolic L. monocytogenes. And if so, do you know whether this is a TAP-dependent process? We wonder whether, in addition to the MHC Ibrestricted CD8 T cells, conventional MHC class-I restricted CD8 T cells could also be activated by antigens originating from the endosome, because alternative pathways have been described from the endosome to the conventional MHC class I processing pathway. Such an assumption would be consistent with earlier findings.
Reply from Kurlander “We clearly must acknowledge the seminal early contribution of Dr. Kaufmann and his collaborators in recognizing ‘MHC-unrestricted’ CTL recognition of L. monocytogenes. It is still unclear how HAA/lemA is processed and presented. New H2M3VVZ biosynthesis is clearly required, and both we (unpublished data) and others (Lenz et al., 19961 have noted that presentation is greatly diminished but not absolutely blocked in Tap2-deficient animals. Simple shunting of antigen from endosome to cytoplasm clearly is not sufficient for initiation of processing and presentation; hence, we believe processing proceeds either by late shunting (i.e. after several hours) of endosomally preprocessed antigen into the cytoplasm for endogenous processing or by a novel pathway of antigenic peptide binding to H2M3ti within the endosome. Additional studies will be required to distinguish between these possibilities.”
On White et al. The article by White et al. reviews the importance of CD8 T cells in murine infection by L. monocytogenes/S. typhimuriun&f. bovis BCG or M. tubercuiosis. The in vivo relevance of CD8 T cells is still questionable in murine S. typhimurium infection. Nevertheless, we have evidence that attenuated S. typhimurium equipped with phagolysosomal escape function did not alter the course of infection in &mdeficient mice in comparison with the control strain (Hess et al., J. Immunol., 156, 3321-3326, 1996). These data suggest that, even in this case, CD8 T cells were not required for Salmonella clearance. As a corollary, we think that clearance of primary S. typhimurium infection only requires a CD4 T-cellmediated immune response. In secondary Sulmonella
BACTERIA
609
ssp. infection, the number of stimulated CD8 T cells increases (Pope et al., Immunology, 81, 177-182, 1994) but the relevance to host defence still has to be clarified.
Reply from White et al. “The experimental results of Hess and Kaufmann demonstrating that phagosomal escape did not enhance Salmonella virulence in pZrn knockouts are consistent with the interpretation that clearance of primary Salmonella infection is independent of CD8+ T cells (Hess et al., J. Immuno/., 156, 3321-3326, 1996). However, these recombinant Salmonella lack the actin-polymerizing activity and intracellular spread capability that are essential virulence determinants of Listeria and Shigella infection. Thus, cytoplasmic Salmonella are still trapped within the initially infected cell. The infected cells will eventually die in a fashion that is independent of the activities of CD8’ T cells, although CD8+ T-cell killing could result in destruction of the infected cell prior to extensive replication of Salmonella.”
On North and Medina We consider it highly interesting that resistance/susceptibility to lung tuberculosis does not follow that to BCG in different mouse strains. Do you know whether genes have been identified in regions near the Nramp gene which could cosegregate in BALB/c versus DBA mice? Such an impact of cosegregating genes could be avoided by using Nramp gene knockout mice which have been generated by Vidal et al. It is apparent to us that the data presented clearly show that the difference is related to the lung and knowledge about potential candidate genes which might interfere with Nramp in control of lung tuberculosis would be an interesting line of future investigation.
Reply from North and Medina “We agree that it would be interesting to look at resistant mice in which Nrampl has been functionally deleted by homologous recombination. However, the results of our study of 100 F progeny of BALB/c (Nramp1 sus) and DBAII (NramplfeSl mice show that Nramp7 segregates independently of resistance to M. tuberculosis, in that heterozygous F mice, as well as F, mice homozygous for each Nramp1 allele, died randomly between 80 and 400 days after receiving IO5 M. tuberculosis i.v. This would argue against
610
67th FORUM
linkage to Nramp7, ready for surprises.”
although
we are always
IN IMMUNOLOGY On Brombacher
and Kopf
Since performing the research discussed in this article, we have become aware of an earlier paper by Musa et al. (Infect. Immun., 55, 1863-1866, 1987) showing that mice now known to be homozygous for the NramplreS allele are not more resistant to h4. tuberculosis infection than mice homozygous for the NramplSUS allele, according to bacterial growth in the lung, liver and spleen over 50 days of infection. Although congenic mice were not used in the study, the results leave little doubt that the role of Nrampl in anti-tuberculosis resistance is questionable. We apologize for having missed this paper. It is apparent that it has escaped the notice of most people active in the field.
I enjoyed reading your paper, particularly the issue that either IL10 or IL4 may act as counterregulators of IFNy-mediated response, depending on the pathogen and the resulting host pathology and immunity. Similarly, in Candida albicans murine infections, we have seen that either IL4 or IL10 may oppose the lL12-dependent Thl response, depending on the yeast/host combination and the ensuing immunopathology. In the case of Listeria, ILlO, more than lL4, appears to act as a counterregulatory cytokine in infection, as the data on KO mice suggest. However, I was wondering whether IL4 and/or IL10 could be produced in mice highly susceptible to infection, such as in STAT-l-‘or IFNy-‘or IFNyR-‘mice. Is the susceptibility of these mutant mice reversed by IL4 or IL10 neutralization? And, in either case, could the result be related to the type of infection ?
Commentary
Reply from Brombacher
General commentary
by R. North and E. Medina:
by L. Romani :
On Hess and Kaujinann I find your results on Salmonella enterica infection very important, particularly with respect to the differences between the different intracellular pathogens. One most impressive finding appears to be the very efficient bacterial control observed in the first 20 days of infection that was clearly IFNydependent and T-cell-independent. However, TCRP-‘- mice efficiently control the infection at 20 days postinfection, at a time when IFNy-deficient mice succumb to infection. It appears that other alternative cellular sources of IFNy are at work or additional mechanisms controlling infection are operative. Would you like to comment on the early phase control of infection?
Reply from Hess and Kaufmann “We think that in the early phase (until day 20 p.i.) NK ceils are the dominant contributors to the control of Salmonella enterica infection. Consistent with this notion is the course of salmonellosis in Ap-Iand IFNyR-‘--deficient mutants. After day 20 p.i., the mutant mice suffer from severe chronic infection or die of salmonellosis, respectively. In addition, RAG-l-/animals which are devoid of functional T and B lymphocytes have a longer survival time than IFNyR-I- mice infected with S. enterica. These data suggest that NK cells may control Salmonella infection via IFNy secretion and/or NK activity.”
“We have no data on STAT-l-/or IFNy-Imice, but we recently et-formed Listeria infection studies in IFNyR- P- IL4-/- double-deficient mice. Two experiments (5 mice/group) with low dose infection (around 200 CFU) were performed by us. In the first experiment, all double-deficient mice died on the same day as IFNyR deficient mice. In the second experiment, most double-deficient mice died with a delay of one day, but one survived. We now control whether the surviving mouse has immunity to a secondary response. However, in a recently published study by S. Kaufmann’s group using anti-ILCtreated IFN$V/- mice, all mice survived and cleared bacteria after a similar low infection dose. At the moment, we cannot explain why anti-114 treatment has stronger effects, but, taken together, these data indicate that depletion of endogenous IL4 has at least some protective influence in IFNyR-‘- mice. The cellular source for the increased resistance has to be identified and may have influenced innate and specific responses. The second part of your question, does IL10 neutralization reverse the phenotype in IFNyR-I- mice, is also very interestin We are in the process of establishing ILIO- 3.- IFNyR-I- double-deficient mice and we hope to answer this question. Nevertheless, as extracted from this Forum showing that infection with a certain pathogen may activate or suppress particular immunoregulatory cell types (and it seems we are finding more and more of them), I do not doubt that the answers we are getting are indeed related to the type of infection.”