The Th1-derived cytokine IFN-gamma inhibits the proliferation of Th2 lymphocytes, but the mechanism of inhibition is not known. Under certain disease conditions, an established Th2-mediated immune response is undesirable and a Th1-mediated response is beneficial. However, established Th2 cells appear to be phenotypically stable. Thus, learning more about cytokine-mediated regulation of established Th2 cells is important if deleterious immune responses are to be altered. We studied the effects of IFN-gamma on a panel of recently derived Th2 lines and clones, as well as a previously established Th2 clone, 13.26. Inhibition by IFN-gamma was observed only when there was a concomitant response to IL-1, a known costimulator of Th2. Clone 13.26 was particularly sensitive to both IL-1 and IFN-gamma, so it was studied in greater detail. We examined cytokine responses using stimulation by anti-TCR mAb-coated plates, or Ag presented by APC populations that do or do not produce IL-1. All IL-1-mediated proliferative responses of 13.26 were inhibited by IFN-gamma, whereas IL-1-independent (IL-4-associated) responses were unaffected. Our data suggest that IFN-gamma inhibits Th2 proliferation through an IL-1-dependent mechanism, and furthermore, that the costimulatory pathways used by APCs may be critical for subsequent Th cell responses to cytokines.
In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2k, I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and Fc gamma RII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-Ek) and the B7 antigens (counter-receptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC.
Abstract Macrophages adopt an ‘alternative phenotype’ in a Th2-mediated inflammatory environment. Although identified in a wide variety of disease conditions, alternatively activated macrophages (AAMs) have been functionally best studied in helminth infections. The heterogeneity of functions performed by AAMs is attributed to the diverse molecules they express. Since infection with the fungus, Aspergillus fumigatus induces a chronic allergic response in patients with cystic fibrosis and asthma, we investigated the participation of AAMs in the process. We have found that Aspergillus infection promotes early recruitment of AAMs to the lung and the cells possess features of both AAMs and Th2 cells. The AAMs produce Th2 cytokines, which is independent of Dectin-1 signaling. Interestingly, unlike in helminth infections, both AAM recruitment and Th2 cytokine production were found to be independent of IL-4Rα and STAT6. Besides being part of the innate immune response to A.fumigatus, AAMs were also induced during adaptive immune response to the fungus. However, at this later time, AAM recruitment and their typical features were abolished in the absence of IL-4Rα and STAT6. Collectively, our studies show that different signaling pathways regulate the development of AAMs in the innate and adaptive arms of the immune response to A. fumigatus. Being early producers of Th2 cytokines, the presence of AAMs would be deleterious to the host unless regulated efficiently.
Abstract The B7 costimulatory family member B7-DC is expressed on activated, but not resting DCs. It is known to inhibit T cells via binding to PD-1, but in PD-1-deficient animals it reportedly delivers a stimulatory signal via an unidentified receptor. We found that it is differentially expressed on lung lymph node DCs from animals undergoing airway inflammation, and examined the functional consequences of this. One day after 3 daily intranasal immunizations, we observed that a majority of lung lymph node myeloid DCs expressed B7-DC. However, expression rapidly diminished only to be re-expressed by 5 days post-immunization. DCs sorted based upon B7-DC expression 1 day following immunization enhanced T cell cytokine production compared to DCs lacking expression, whereas those isolated 5 days post-immunization stimulated less cytokine production. This effect was evident will all three types of T helper cells (Th1, Th2, and Th17) using several different antigen/adjuvant systems. The results could not entirely be explained by differential expression of PD-1 since similar data was obtained with naïve T cells, which do not initially express PD-1, and with activated T cells, which do. These data support a dual role for B7-DC and further suggest that mature myeloid-type DCs can be both stimulatory or inhibitory based upon B7-DC expression. (RO1 HL77430 to A.R. and RO1 HL60207 to P.R.)
Abstract Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology in which T cell responses to various autoantigens, including DNA topoisomerase I (Topo I), have been implicated. We investigated whether dendritic cells, generally considered to be the most potent APCs for the initiation of immune responses, would present either of two forms of Topo I to T cells more efficiently than PBMC APCs. Using cells from healthy controls and SSc patients, several important observations were made. First, neither APC type was able to initiate T cell proliferative responses to full-length native Topo I unless exogenous IL-2 was added. This is in contrast to vigorous T cell proliferation in response to Topo I polypeptide fragments presented by either APC type. Second, T cell responses to the full-length form of Topo I presented by dendritic cells were considerably lower than responses to Ag presented by PBMC APCs. Finally, no secondary T cell responses were observed unless the same Ag/APC combination as that used in the primary stimulation was maintained. These data indicate that different peptides are generated based upon the form of the Topo I and the APC that processes it. Taken together, these results suggest that a very specific combination of antigenic form and APC may be involved in breaking tolerance to Topo I in the early stages of development of SSc.
Severe asthma (SA) is a significant problem both clinically and economically, given its poor response to corticosteroids (CS). We recently reported a complex type 1-dominated (IFN-γ-dominated) immune response in more than 50% of severe asthmatics despite high-dose CS treatment. Also, IFN-γ was found to be critical for increased airway hyperreactivity (AHR) in our model of SA. The transcription factor IRF5 expressed in M1 macrophages can induce a Th1/Th17 response in cocultured human T cells. Here we show markedly higher expression of IRF5 in bronchoalveolar lavage (BAL) cells of severe asthmatics as compared with that in cells from milder asthmatics or healthy controls. Using our SA mouse model, we demonstrate that lack of IRF5 in lymph node migratory DCs severely limits their ability to stimulate the generation of IFN-γ- and IL-17-producing CD4+ T cells and IRF5-/- mice subjected to the SA model displayed significantly lower IFN-γ and IL-17 responses, albeit showing a reciprocal increase in Th2 response. However, the absence of IRF5 rendered the mice responsive to CS with suppression of the heightened Th2 response. These data support the notion that IRF5 inhibition in combination with CS may be a viable approach to manage disease in a subset of severe asthmatics.
