H. Kirk Ziegler

Paradoxical Anti-Inflammatory Actions of TNF-α: Inhibition of IL-12 and IL-23 via TNF Receptor 1 in Macrophages and Dendritic Cells

IL-12 and TNF-α are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-α is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-α. TNF-α-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-γ and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-α in TNFR1−/−-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-α than cells from naive animals, which suggests a regulatory role for TNF-α in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-α-induced responses determined by cell-specific receptor signaling.

Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential requirements for the processing and presentation of soluble and particulate bacterial antigens by macrophages

The requirements for antigen processing and presentation by macrophages using various forms of antigens derived from Listeria monocytogenes have been studied. Antigen presentation was monitored by T cell‐macrophage binding and interleukin production using T cells from Listeria monocytogenes‐infected mice and specific T cell hybridomas. Antigen processing requirements were defined by three criteria: (a) inhibition by lysosomotropic agents, NH4Cl and chloroquine; (b) kinetic relationships between antigen uptake and antigen presentation; and (c) antigen presentation by macrophages pre‐fixed with glutaraldehyde. In comparing heat‐killed Listeria monocytogenes (HKLM) with soluble listerial proteins (SLP), the presentation of SLP was less sensitive to lysosomotropic agents, showed faster antigen processing kinetics than with HKLM and could occur using pre‐fixed macrophages. Transitions between particulate and soluble forms had dramatic influences on processing requirements. Antigens associated with HKLM could be converted to soluble forms which did not require processing by preculture with macrophages and also by physical (e.g. sonication) and chemical (sodium dodecyl sulfate) treatments in the presence of protease inhibitors. Conversely, antigen processing was required when SLP were converted to a particulate form by covalent binding to latex beads. Analysis of SLP by molecular sieve chromatography and preparative SDS‐polyacrylamide gel electrophoresis revealed that high molecular weight proteins (> 60 kDa) could be presented by prefixed macrophages without prior processing. We conclude that the transition from a particulate to soluble antigenic form can be a significant antigen processing event.

Exaggerated proinflammatory and Th1 responses in the absence of gamma/delta T cells after infection with Listeria monocytogenes.

ABSTRACT While γ/δ T cells are involved in host defense and immunopathology in a variety of infectious diseases, their precise role is not yet clearly defined. In the absence of γ/δ T cells, mice die after infection with a dose of Listeriamonocytogenes that is not lethal in immunologically intact animals. Morbidity might result from insufficient levels of cytokines normally produced by γ/δ T cells or conversely from an excess of cytokines due to a lack of down-regulation of the inflammatory response in the absence of γ/δ T cells. Consistent with a regulatory role, we found that systemic levels of proinflammatory cytokines (interleukin-6 [IL-6], IL-12, and gamma interferon [IFN-γ]) were significantly higher in the absence of γ/δ T cells during the innate phase of the response. Using combinations of genetically altered and immunodepleted mice, we found evidence for γ/δ T-cell-mediated regulation of IFN-γ production by multiple cell types of both lymphoid and myeloid lineages. The antigen-specific α/β T-cell response that followed the exaggerated innate response was also increased in γ/δ T-cell-deficient mice. These findings are consistent with an emerging picture from a variety of immune response models of a critical role for γ/δ T cells in down-modulation of the immune response.

Changes in peritoneal myeloid populations and their proinflammatory cytokine expression during infection with Listeria monocytogenes are altered in the absence of γ/δ T cells

Evidence that γ/δ T cells play a broad, immunoregulatory role has been accumulating steadily. We show here that myeloid cells are disregulated after peritoneal infection with Listeria monocytogenes in mice lacking γ/δ T cells. Inflammatory populations of neutrophils and monocytes recruited to the site of infection remained longer. Intracellular cytokine analysis showed that frequencies of myeloid cells producing interleukin‐12 and tumor necrosis factor α were higher and remained elevated longer after infection in mice genetically deficient in γ/δ T cells. In vivo dye‐tracking studies indicated that the majority of inflammatory monocytes differentiated into resident tissue macrophages in situ. In vitro experiments confirmed that monocytes harvested from mice lacking γ/δ T cells were defective in their maturation process. This evidence suggests that γ/δ T cells promote differentiation in the monocyte/macrophage lineage. These cells are important for bactericidal activity, inflammatory cytokine production, clearance of inflammatory neutrophils, and ultimately, antigen presentation to T cells. Regulation of monocyte/macrophage differentiation may underlie a broad segment of the phenotypic alterations that have been reported in mice lacking γ/δ T cells.

Protective immunity to Listeria monocytogenes elicited by immunization with heat-killed Listeria and IL-12. Potential mechanism of IL-12 adjuvanticity

The results presented here demonstrate the striking potentiating effects of IL-12 when it is combined with listerial immunogens. Although HKLM alone does not elicit strong T-cell responses, the results presented here demonstrate that the combination of HKLM and IL-12 elicited vigorous Listeria-specific Th1-type T-cell responses when administered intraperitoneally. The intensity of these responses, as well as the cytokine profiles of the Listeria-specific peritoneal T cells and macrophages, was remarkably similar to that of Listeria-infected/immune mice. These studies also revealed that typically nonimmunogenic forms of soluble listerial antigen preparations (cLLO, SLP) and LLO peptide homologs (M. A. Miller et al., manuscript in preparation) elicited intense Listeria-specific T-cell responses when administered with IL-12. In conjunction with the generation of specific T-cell responses following injection of IL-12 in combination with either killed Listeria or soluble listerial antigen preparations, macrophages from these mice expressed upregulated quantities of class II MHC and produced increased amounts of IL-12 following restimulation in vitro. Protection studies established that the Listeria-specific T-cell responses elicited by the HKLM + IL-12 mixture conferred protective immunity of mice to a lethal dose of viable L. monocytogenes. Studies designed to investigate the regulation of IL-12 production by peritoneal macrophages revealed that activated macrophages are particularly sensitive to bacterial products. However, nonviable or replication-incompetent bacteria or bacterial products injected alone were unable to influence the ability of macrophages to produce IL-12. The ability of activated macrophages to respond to HKLM was dramatically upregulated upon addition of IFN-gamma and markedly downregulated in the presence of the Th2 cytokines, IL-4 and IL-10. In light of what is known about the ability of IL-12 to induce IFN-gamma production by NK cells and gamma delta T cells, these results suggest that the exogenous addition of IL-12 may help initiate a cytokine cascade which enables the immune system to interact productively with an antigen that is typically nonimmunogenic when administered alone. These findings demonstrate that IL-12 may prove to be a powerful and broadly useful adjuvant component of particulate and soluble antigen-based vaccines directed towards many types of intracellular pathogenic microorganisms. Studies aimed at determining the generality of these findings in other infectious disease models as well as experiments designed to further elucidate the mechanism(s) of IL-12 adjuvanticity are continuing.

The role of γ/δ T cells in immunity to infection and regulation of inflammation

The role of γ/δ T cells in immunity to bacterial infection and control of inflammation is discussed. Special emphasis is placed on the use of murine models in which various aspects of immune function can be monitored in the absence of γ/δ T cells. Issues discussed include the response to and control of cytokine production by γ/δ T cells. Especially relevant is the apparent paradox that γ/δ T cells play both pro-inflammatory and antiinflammatory roles in response to infection.The role of γ/δ T cells in immunity to bacterial infection and control of inflammation is discussed. Special emphasis is placed on the use of murine models in which various aspects of immune function can be monitored in the absence of γ/δ T cells. Issues discussed include the response to and control of cytokine production by γ/δ T cells. Especially relevant is the apparent paradox that γ/δ T cells play both pro-inflammatory and antiinflammatory roles in response to infection.

Role of α/β T and γ/δ T Cells in Innate and Acquired Immunity

T-lymphocyte subpopulations can be classified according to the differential expression of T-cell receptors, surface markers, adhesion molecules, cytokines, and their receptors. One distinction is in the expression of T-cell surface molecules such as CD4 and CD8. The binding of CD4 to class I1 MHC molecules or CD8 to class I MHC molecules is thought to create a requisite stabilizing “bridge” between the antigenpresenting cell and the responding T cell as well as a conduit of transducing signals. Class I MHC molecules displayed on a variety of cells are involved in antigen presentation to CD8’ T cells, whereas class I1 MHC molecules, expressed on a more restricted spectrum of cell types such as macrophages and B cells, serve as recognition molecules for CD4+ T cells. As such, the appropriate interactions of CD8’ cytotoxic T cells with infected cells and CD4’ helper cells with B cells and macrophages are facilitated. T cells of the CD4’ subset can also be subdivided into at least two major types by their patterns of cytokine production. TH1 cells secrete gamma-interferon (INF-y) and interleukin-2 (IL-2), whereas TH2 cells produce IL-4, IL-5, IL-6, and IL10. These differences in lymphokine secretion correlate with functional specialization. TH1 cells are involved in delayed-type hypersensitivity reactions and macrophage activation: whereas TH2 cells may be more important for helping B cells respond with expression of certain antibody isotypes.4 Differential regulation of these subsets plays an important role in infectious disease,’ transplantation reactions: and hypersensitivity .7

Simultaneous Th1-Type Cytokine Expression Is a Signature of Peritoneal CD4+ Lymphocytes Responding to Infection with Listeria monocytogenes

The robust murine response to infection with Listeria monocytogenes makes an excellent model to study the functional development of immune cells. We investigated the cellular immune response to i.p. infection using intracellular cytokine staining to identify Ag-specific lymphocytes. CD4+ peritoneal exudate cells obtained 10 days postinfection predominantly coexpressed TNF-α, IFN-γ, and IL-2 after polyclonal or Ag stimulation. A population of cells simultaneously making TNF-α and IFN-γ was also detected but at a lower frequency. By following the kinetics of the response to Listeria, we found that CD4+ lymphocytes coexpressing TNF-α and IFN-γ dominated on day 6 postinfection and then declined. From days 10–27, TNF-α+IFN-γ+IL-2+ (triple-positive) was the most prevalent cytokine phenotype, and the frequency steadily declined. These characteristic cytokine expression patterns were observed in both primary and secondary responses to Listeria infection and developed even when infection was terminated with antibiotic treatment. A cytokine-assisted immunization procedure resulted in both double- and triple-positive cells, but the clear predominance of triple-positive cells required Listeria infection. Triple-positive cells were preferentially noted in the peritoneal cavity tissue site; spleen cells displayed a predominant population of double-positive T cells (TNF-α+IFN-γ+). We speculate that the appearance of triple-positive cells represents a functionally significant subset important in host defense at nonlymphoid tissue sites.

IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes

Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity. We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12. Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system. We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization. IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses. In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response. IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed.