Ex-vivo tolerogenic F4/80⁺ antigen-presenting cells (APC) induce efferent CD8⁺ regulatory T cell-dependent suppression of experimental autoimmune uveitis.

It is known that inoculation of antigen into the anterior chamber (a.c.) of a mouse eye induces a.c.-associated immune deviation (ACAID), which is mediated in part by antigen-specific local and peripheral tolerance to the inciting antigen. ACAID can also be induced in vivo by intravenous (i.v.) inoculation of ex-vivo-generated tolerogenic antigen-presenting cells (TolAPC). The purpose of this study was to test if in-vitro-generated retinal antigen-pulsed TolAPC suppressed established experimental autoimmune uveitis (EAU). Retinal antigen-pulsed TolAPC were injected i.v. into mice 7 days post-induction of EAU. We observed that retinal antigen-pulsed TolAPC suppressed the incidence and severity of the clinical expression of EAU and reduced the expression of associated inflammatory cytokines. Moreover, extract of whole retina efficiently replaced interphotoreceptor retinoid-binding protein (IRBP) in the preparation of TolAPC used to induce tolerance in EAU mice. Finally, the suppression of EAU could be transferred to a new set of EAU mice with CD8⁺ but not with CD4⁺ regulatory T cells (T(reg)). Retinal antigen-pulsed TolAPC suppressed ongoing EAU by inducing CD8⁺ T(reg) cells that, in turn, suppressed the effector activity of the IRBP-specific T cells and altered the clinical symptoms of autoimmune inflammation in the eye. The ability to use retinal extract for the antigen raises the possibility that retinal extract could be used to produce autologous TolAPC and then used as therapy in human uveitis.

Ly49 C/I-dependent NKT cell-derived IL-10 is required for corneal graft survival and peripheral tolerance.

Similar to their activity on NK cells, Ly49 molecules play a pivotal role in influencing how NKT cells respond. It is known that Ly49 C/I is an inhibitory receptor capable of down-modulating proliferation, IFN-gamma response, and cytotoxic activity in cells that express it. In a model of peripheral tolerance induced via the eye, we observed that Ly49 C/I-positive, invariant NKT cells were required. To test if the NK inhibitory receptor functionally contributed to tolerance development, we used blocking antibody, in vivo and in vitro, to interfere with the development of antigen-specific suppression. A result of blocking ligation of Ly49 C/I inhibitory receptor prevented NKT cell production of IL-10 and the subsequent development of tolerance. Ly49 C/I-blocking antibodies also prevented corneal graft survival, a phenomenon dependent on eye-induced tolerance. Furthermore, in the presence of TCR stimulation, cross-linking of Ly49 C/I on CD4(+) NKT cells stimulated an increase in IL-10 mRNA and a decrease in IFN-gamma. The concept of Ly49 inhibitory receptors regulating immune reactivity to self by regulating immune activity of individual cells is thus expanded to include a role for the inhibitory receptors in the more global process of peripheral tolerance to foreign antigens.

The transcription factor early growth-response factor 1 modulates tumor necrosis factor-alpha, immunoglobulin E, and airway responsiveness in mice.

Early growth-response factor 1 (Egr-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important in inflammation, cell growth, apoptosis, and the pathogenesis of disease. In vitro studies suggest that Egr-1 is capable of regulating the expression of tumor necrosis factor-alpha (TNF-alpha) and other genes involved in airway inflammation and reactivity following allergen stimulation. On the basis of these data, we hypothesized that in the absence of Egr-1, the TNF-alpha response and subsequent downstream inflammatory events that usually follow allergen challenge would be diminished. To test our hypothesis Egr-1 knock-out (KO) mice were examined in an ovalbumin (OVA)-induced model of airway inflammation and reactivity, and compared with identically treated wild-type (WT) control mice. In response to OVA sensitization and airway challenge, KO mice had diminished TNF-alpha mRNA and protein in the lungs and mast cells compared with WT mice. Interestingly, the KO mice had elevated IgE levels at baseline and after allergen challenge compared with WT mice. Furthermore, the airways of KO mice were hyporesponsive to methacholine challenge at baseline and after allergen challenge. These data indicate that Egr-1 modulates TNF-alpha, IgE, and airway responsiveness in mice.

CD40/CD40 ligand interactions are critical for elicitation of autoimmune-mediated fibrosis in the lung.

Pulmonary interstitial fibrosis (PIF), associated with persistent inflammation and increased collagen deposition in the interstitium, is often considered an autoimmune disease. Hapten immune PIF (HIPIF), a model for PIF, is elicited in the lung by a single intratracheal (i.t.) challenge in mice sensitized with hapten (2,4,6-trinitrobenzene sulfonic acid, TNBS). In this study, we characterized the role of CD40/CD40 ligand (CD40L) interactions in the elicitation of secondary cell-mediated immune responses that lead to development of fibrosis in the lung using an adoptive transfer model of HIPIF. The expression of CD40 was detected on bronchoalveolar lavage (BAL) cells 1-3 days after i.t. challenge with hapten in the HIPIF lung, but not lungs from the control mice. The CD40(bright) BAL cells morphologically resembled infiltrating monocytes. Furthermore, blocking CD40/CD40L interactions with blocking Ab decreased BAL production of Th1-mediators (IL-12 and TNF-alpha). Moreover, either blocking CD40/CD40L interactions with the Ab or using IL-12 knockout recipient mice prevented the increased collagen deposition (accumulation of hydroxyproline) in the lungs during HIPIF induction. We conclude that second signals (CD40/CD40L interactions) are required for elicitation of secondary immune responses that lead to PIF in vivo. The results support the notion that CD40/CD40L interactions are involved in the pathogenesis of an ongoing autoimmune disease.

NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance.

In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant V alpha 14J alpha 281 TCR because J alpha 281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in J alpha 281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.

MIP-2 recruits NKT cells to the spleen during tolerance induction.

Peripheral tolerance occurs after intraocular administration of Ag and is dependent on an increase in splenic NKT cells. New data here show that macrophage inflammatory protein-2 (MIP-2) is selectively up-regulated in tolerance-conferring APCs and serves to recruit NKT cells to the splenic marginal zone, where they form clusters with APCs and T cells. In the absence of the high-affinity receptor for MIP-2 (as in CXCR2-deficient mice) or in the presence of a blocking Ab to MIP-2, peripheral tolerance is prevented, and Ag-specific T regulatory cells are not generated. Understanding the regulation of lymphocyte traffic during tolerance induction may lead to novel therapies for autoimmunity, graft acceptance, and tumor rejection.

A critical role for alveolar macrophages in elicitation of pulmonary immune fibrosis.

Hapten immune pulmonary interstitial fibrosis (HIPIF) is induced by a recall cell-mediated immune response against the hapten 2,4, 6-trinitrobenzene sulphonic acid (TNBS) in the lung. Studies here dissect the role of the cellular components of the bronchoalveolar lavage (BAL) cells (alveolar macrophages [AMs] versus monocytes and immature dendritic cells) in the fibrogenic inflammatory response. BAL cells from HIPIF mice were generally more activated and produced a greater amount of tumour necrosis factor-alpha (TNF-alpha) than controls. Liposome-encapsulated dichloromethylene diphosphonate (Cl(2)MDP) that was inoculated intranasally (i.n.) into mice selectively depleted AMs. Following AM depletion, the number of TNF-alpha-containing cells was reduced, and both the number of immune inflammatory cells recruited into the alveolar space and the subsequent collagen deposition (hydroxyproline) were decreased in the sensitized and intratracheally (i.t.) challenged mice. In conclusion, AMs are required, in part, for the development of pulmonary fibrosis in HIPIF because AM-derived factors such as TNF-alpha are needed for initiation of chemokine and cytokine pathways and accumulation of immune inflammatory cells.

Does innate immune privilege exist?

Immune privilege in the eye is believed to originate from the eye's need to avoid the sight-destroying consequences of inflammation. Over the past 25 years, many of the anatomical, cellular, and molecular mechanisms by which the eye avoids inflammation secondary to adaptive immune responses have been elucidated. In the recent past, it has become increasingly clear that innate immune responses play a critical role in activating the adaptive immune response. Moreover, innate immunity itself carries a heavy burden of inflammation, thereby posing a threat to vision if it should occur intraocularly. Ocular immunologists have now begun to inquire into the extent to which the eye regulates the expression of innate immunity in oculi. Evidence is presented which indicates that factors found in normal aqueous humor (1) prevent NK cells from lysing their targets, (2) inhibit neutrophil activation by CD95 ligand, (3) suppress nitric oxide production by activated macrophages, and (4) interfere with complement activation via the alternative pathway. These factors include transforming growth factor-beta2, alpha-melanocyte-stimulating hormone, calcitonin gene-related peptide, and migration inhibitory factor. The ability of the eye to prevent intraocular activation of innate immune effectors spares the corneal endothelium (which expresses CD95 ligand constitutively, but low levels of MHC class I molecules) from destruction by NK cells and neutrophils, and protects the visual axis from distortion by macrophage and complement-mediated inflammation. Thus, privilege exists in the eye for both adaptive and innate immunity.

Regulation of adaptive immune responses by innate cells expressing NK markers and antigen-transporting macrophages.

A continuing theme of work done in our laboratory involves regulation of adaptive immune response by innate cells, in general, and immuneregulation by natural killer (NK) and NKT cells, in particular. Studies include work with the lung and the eye. In addition to immune surveillance of tumor cells, the NK cell is often associated with secreting cytokines that contribute to the creation of microenvironments conducive to Th1 responses and with defense mechanisms that lessen the initial infecting viral load. Reported studies show that the NKT cells support both T helper cell responses (type 1 and 2), as well as their being absolutely central to the development of antigen-specific T-regulatory cells involved in peripheral tolerance. Because of the multifunctional capabilities of the NKT cell, we propose that yet another cell, such as the antigen-presenting cell (APC), may influence the effector pathway of the NKT cell. We postulate that the APC that transports the antigen from the entry environment provides both trafficking and activation signals for innate cells in the secondary lymphoid organs. Evidence is presented that macrophage-derived signals selectively recruit NKT cells and bias their cytokine synthesis. Data imply that, just as occurs in immune inflammation, a collection of innate and adaptive immune cells interact within the secondary lymphoid tissue to generate antigen-specific tolerance in the periphery.

CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site.

Systemic tolerance can be elicited by introducing antigen into an immune-privileged site, such as the eye, or directly into the blood. Both routes of immunization result in a selective deficiency of systemic delayed type hypersensitivity. Although the experimental animal model of anterior chamber-associated immune deviation (ACAID) occurs in most mouse strains, ACAID cannot be induced in several mutant mouse strains that are coincidentally deficient in natural killer T (NKT) cells. Therefore, this model for immune-privileged site-mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance. The following data show that CD1-reactive NKT cells are required for the development of systemic tolerance induced via the eye as follows: (a) CD1 knockout mice were unable to develop ACAID unless they were reconstituted with NKT cells together with CD1(+) antigen-presenting cells; (b) specific antibody depletion of NKT cells in vivo abrogated the development of ACAID; and (c) anti-CD1 monoclonal antibody treatment of wild-type mice prevented ACAID development. Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes. A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.

Interleukin-8 receptor modulates IgE production and B-cell expansion and trafficking in allergen-induced pulmonary inflammation.

We examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r-/-). Wild-type (Wt) and IL-8r-/- mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r-/- mice compared with Wt mice, whereas multiply challenged IL-8r-/- mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r-/- OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r-/- OVA/OVA mice than in Wt mice. Both the IL-8r-/- OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.

Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4.

In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti-CD3 and anti-CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non-specific mechanism. C57BL/6 mice were inoculated with anti-natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T-cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin-4 (IL-4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T-cell deficient [beta 2-microglobulin beta 2m-deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when beta 2m-deficient mice were reconstituted with NK T cells. NK-specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL-4.

Effects of treatment with pentoxifylline on the cardiovascular manifestations of group B streptococcal sepsis in the piglet.

Pentoxifylline (PTXF) is a methylxanthine derivative which modifies leukocyte function and inhibits tumor necrosis factor (TNF)-alpha release. As TNF-alpha is considered a proximal mediator in the cascade leading to septic shock, we evaluated the ability of PTXF to attenuate the cardiovascular manifestations of sepsis secondary to an infusion of group B beta-hemolytic streptococci (GBS). Fifteen anesthetized, mechanically ventilated piglets (weight, 2815 +/- 552 g) were randomly assigned to a treatment group which received a continuous infusion of PTXF (5 mg/kg/h) beginning 30 min after GBS (7.5 x 10(8) colony-forming units/kg/min) administration was started or to a control group which received GBS plus saline as placebo. Comparison of the hemodynamic measurements and arterial blood gases over the first 120 min of bacterial infusion for treatment and control groups revealed the following statistically significant differences (120-min values presented): cardiac output was significantly higher in the PTXF group (0.159 +/- 0.035 versus 0.09 +/- 0.026 L/kg/min; p < 0.05) as was stroke volume (0.54 +/- 0.11 versus 0.27 +/- 0.126 mL/kg/beat; p < 0.01). Pulmonary and systemic vascular resistances remained lower in the PTXF-treated animals (167 +/- 45 versus 233 +/- 69 mm Hg/L/kg/min; p < 0.03) and (427 +/- 162 versus 828 +/- 426 mm Hg/L/kg/min; p < 0.03, respectively). Median survival time was significantly longer in the PTXF group (180 versus 120 min; p < 0.05). In an additional group of animals, PTXF administration before GBS infusion revealed no attenuation in the rise of TNF-alpha, accompanying sepsis. These data demonstrate that treatment with PTXF may ameliorate some of the deleterious hemodynamic manifestations of GBS sepsis and result in improved survival in a young animal model without significantly modifying plasma TNF-alpha levels.

Human natural killer (NK) cells present staphylococcal enterotoxin B (SEB) to T lymphocytes.

Superantigen-mediated T cell activation requires the participation of antigen-presenting cells (APC). Once superantigen has bound class II MHC molecules on the surface of APC, it then can interact with the T cell receptor to induce T cell activation. Superantigen-mediated T lymphocyte activation, along with its consequent cytokine production is thought to be the basis for the pathophysiology of conditions such as toxic shock syndrome, Kawasaki's disease and possibly rheumatoid arthritis. We examined the role of CD56+ NK lymphocytes in the interaction between superantigens and T lymphocytes. First, we found that a subpopulation of CD56+ cells freshly isolated from human peripheral blood expressed class II MHC molecules. The amount of HLA-DR expression varied between individuals, ranging from 9.3% to 37.7%. CD56+ (NK) cells were purified from the peripheral blood by cell sorting and were tested for their ability to support SEB-mediated T cell activation as assessed by surface expression of IL-2 receptor alpha-chain (CD25) on CD3+ lymphocytes. We observed that when enriched T cells were incubated with SEB in the presence of NK cells, there was a significant up-regulation of CD25 expression of the T cells. When HLA-DR+ cells were removed from sorted CD56+ populations, the remaining HLA-DR- NK cells were unable to support SEB-mediated T cell activation. Also, SEB up-regulated the expression of HLA-DR on CD56+ cells in peripheral blood mononuclear cell (PBMC) populations after 24 h of incubation, implying that the ability of NK cells to function as superantigen-presenting cells is up-regulated by superantigens themselves. Together, these data demonstrate for the first time that human CD56+ HLA-DR+ NK cells can function as superantigen-presenting cells, and imply that NK cells may be involved in the activation of non-specific T cell reactivity during early host defences against superantigen-elaborating microorganisms in vivo. Furthermore, the physical linkage of NK cells and T cells by the interaction of superantigen with HLA class II molecules and T cell receptors, respectively, may lead to NK cell activation and augmented lytic potential, helping to clear the body of superantigen-elaborating microorganisms.

Mycoplasma arthritidis mitogen up-regulates human NK cell activity.

While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in both adherent and nonadherent subpopulations. Experiments using cytokine-specific neutralizing antibodies showed that interleukin-2 contributed to enhancement of the NK activity observed in superantigen-stimulated PBMC. Interestingly, MAM was able to augment NK lysis of highly purified NK (CD56+) cells in the absence of other immune cells in 9 of 12 blood specimens, with the augmented lytic activity ranging from 110 to 170% of unstimulated NK activity. In summary, data presented in this report show for the first time that MAM affects human NK cells directly by increasing their lytic capacity and indirectly in PBMC as a consequence of cytokines produced by T cells. Results of this work suggest that, in vivo, one consequence of interaction with superantigen-secreting microorganisms may be up-regulation of NK lytic activity. These findings may have clinical application as a means of generating augmented NK effector cells useful in the immunotherapy of parasitic infections or neoplasms.

Staphylococcal enterotoxin B activates purified NK cells to secrete IFN-gamma but requires T lymphocytes to augment NK cytotoxicity.

Staphylococcal enterotoxin B (SEB) is known for its effects on T lymphocytes; the mechanism of this activation is well studied. Although it is anticipated that there would be effects on other cells in the milieu, the mechanisms of activation of non-T immune cells are not well understood. This report examines the effects of SEB on human NK cells. Incubation of PBMC with SEB augmented NK cytotoxicity against the NK-sensitive targets K562 and Molt-4 and induced activity against the NK-resistant targets, Mel-30 and Raji. The degree of superantigen-augmented killer (SAK) activity induced by the SEB treatment correlated with the concentration of the superantigen in the mixture. Specific neutralizing Abs for lymphokines or monokines added to cultures showed that IL-2, IFN-gamma, or IL-12 each participated in the phenomenon; addition of all Abs together blocked most, but not all, SAK activity. Highly purified T lymphocytes (but not monocytes) supported the development of SAK activity in enriched cultures of CD56+ cells. The SAK activity was blocked by specific Abs to IL-2 and IFN-gamma in cocultures of T cells and CD56+ cells. In addition, SEB induced IFN-gamma secretion from sorted CD56+ cells. These data show for the first time that superantigen directly initiated cytokine synthesis in NK cells, and that SAK activity was induced as a consequence of products secreted from activated T lymphocytes. Such a superantigen effect on NK cells would place the NK lymphocyte in a pivotal role in determining the outcome of infection or immunopathology caused by superantigen exposure.

Hapten-immune pulmonary interstitial fibrosis (HIPIF) in mice requires both CD4+ and CD8+ T lymphocytes.

We present a description and analysis of a mouse model for pulmonary interstitial fibrosis that is induced by a specific immune response to a small reactive chemical group called trinitrophenyl. We describe the model, and then we examine the cellular mechanism for the induction of the fibrosis. The specific increase in hydroxyproline reached a peak by day 7 and persisted through day 28 in all animals that were sensitized to and challenged with the hapten. Distinct patterns of fibrosis that were seen histologically correlated with antigenic pretreatment and were dependent on T lymphocytes. We also report that the inflammatory and fibrotic responses could be adoptively transferred with immune lymphocytes but not with immune serum. In vivo administration of anti-CD4 and anti-CD8 monoclonal antibodies to sensitized mice prevented the development of immune-mediated lung inflammation and was effective in reducing hydroxyproline deposition. We conclude that (activated) T lymphocytes contribute to the pathogenesis of pulmonary fibrotic diseases. The possibility arises that haptens in the environment may promote sensitization of individuals via their skin or lungs and cell-mediated immune responses to haptenated antigens within the lung may promote pulmonary fibrosis.

Delayed-type hypersensitivity responses regulate collagen deposition in the lung.

A previous report showed that hamsters immunized by epicutaneous application of 2,4,6-trinitrochloro-1-benzene (TNCB) were susceptible to the development of pulmonary interstitial fibrosis (PIF) if challenged in the lung with the water-soluble form of this hapten 2,4,6-trinitrobenzene sulphonic acid (TNBS). In this study, we investigated the immunological mechanisms that contributed to increased collagen content in the lungs of hapten-immune hamsters after receiving a pulmonary challenge of the sensitizing hapten trinitrophenol (TNP). In order to evaluate the concept that delayed-type hypersensitivity (DTH) reaction modulated their response to TNP in the lung such that it eventuated into PIF, we compared the cutaneous DTH response (48 hr after challenge) with lung collagen deposition (14 days after challenge) in several lines (strains) of hamsters. The inbred LSH strain, was a high responder in the DTH assay to TNP and developed non-resolving PIF in the hapten-immune animals. This is called hapten-immune pulmonary interstitial fibrosis or HIPIF. We also observed that female LSH hamsters were more susceptible to HIPIF induced by TNP than males. On the other hand, age factors influenced DTH and PIF in random-bred LVG hamsters since young hamsters (3 months old) were low responders to TNP and did not develop PIF in the HIPIF model but matured LVG hamsters (retired breeders) possessed DTH reactivity to TNP and subsequently developed PIF. These results suggest that lung collagen deposition in hapten-immune hamster is regulated by T-lymphocyte-mediated immune inflammation (DTH) in the lung and both are dependent on the ability to develop a cutaneous DTH reaction to the hapten. The elucidation of possible mechanisms of DTH-mediated non-granulomatous, non-resolving PIF is important for understanding of the role of environmental chemicals similar in action to haptens in the mediation of skin and lung diseases.