Joan Stein-Streilein

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.

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, Kawasakis 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 α‐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 on 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.

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)-α release. As TNF-α 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 β-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 × 108 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 ± 45versus 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-α, 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-α levels.

Immunological tolerance to hapten prevents subsequent induction of hapten-immune pulmonary interstitial fibrosis (HIPIF).

Pulmonary interstitial fibrosis (PIF) is a morphological term which in part can be defined as accumulation of collagen in the extracellular matrix. Previously we showed that hamsters sensitized with 2,4,6-trinitro-1-chlorobenzene (TNCB) developed PIF 14 days after an intratracheal challenge with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The participation of delayed-type hypersensitivity (DTH) in lung collagen deposition was clearly demonstrated. In this paper, we use an adaptation of this model to mice and show that the lung collagen deposition observed was related to the genetic ability of the strain to maintain a DTH response to the immunizing hapten (TNP). Specifically, the lung collagen deposition on Day 14 in hapten-sensitized, challenged animals in high responder to TNP (BALB/c, H-2d) was higher than that in low responder mouse (C57BL/6, H-2b). Furthermore, aged C57BL/6 strain (retired breeders) possessed a DTH response to TNP and produced significantly higher accumulation of hydroxyproline than that of TNBS-challenged-only animals. A DTH mechanism for the induction of the fibrosis is consistent with the observation that responder mice that were made tolerant to the antigen were unable to respond to the lung challenge with a specific increase in lung index or collagen deposition. These results suggest that effector T lymphocytes that are important in DTH play a key role in the regulation of lung collagen deposition in hapten-immune pulmonary interstitial fibrosis (HIPIF) in mice.

Natural Effector Cells in Influenza Virus Infection

It is known that specific IgA antibody is protective in preventing influenza infection.(1,2) Once infected, however, cellular mechanisms of the host are important and contribute toward limiting the infection.(3) The nature of the cellular immune effectors that develop in mice in response to influenza virus infection has been well described. Cytotoxic T lymphocytes (CTL) play a crucial role in preventing virus spread and dissemination of virus in mice,(4,5) and virus-specific delayed-type hypersensitivity (DTH) T lymphocytes contribute to the destructive intrapulmonary lesion that characterizes influenzal pneumonia disease.(6,7)

Monovalent Fab fragments of D7.5 monoclonal antibody activate intracellular Ca2+ mobilization and secretion of cytolytic factors by thymus cells.

Previous studies have identified two mouse monoclonal antibodies, D7.5 and G1.4, that each can cause secretion of cytolytic factors from natural killer (NK) cells and resting T cells in humans, rats, and hamsters. Toward elucidation of the molecular identity and the transmembrane signaling mechanism of the antibody‐defined trigger molecules, we investigated 1) their surface density, 2) the crosslinking requirement for signaling, and 3) their ability to mobilize intracellular calcium ions. Equilibrium binding studies using monovalent Fab fragments of D7.5 showed the presence of approximately 20,000 trigger molecules per thymus cell and an antibody dissociation constant of 1.8 x 10‐6 M. Thymus cells incubated in the presence of either intact or Fab fragments of D7.5 acquired the ability to lyse Yac‐1 cells. Furthermore, the incubation media of thymocytes that were treated with either intact or Fab fragments of D7.5 contained effector‐cell derived factors that could lyse Yac‐1 cells. Flow cytometry measurements of changes in intracellular free calcium concentration ([Ca2+]i) in thymocytes. A significantly elevated level of [Ca2+]i was observed at 60s after antibody addition and this response, which required the external calcium source, increased continuously during the 30 min incubation. Furthermore, dbcAMP plus theophylline did not alter the ability of D7.5 to induce calcium mobilization suggesting that the antibody‐defined trigger may not use the signaling pathway involving breakdown of phosphatidylinositol.

Monoclonal antibodies (G1.4, D7.5) induce secretion of lytic factors from natural killer and T lymphocytes.

Monoclonal antibodies were made by standard procedures using mononuclear cells isolated from hamster lung tissue to immunize the mice. The Moabs (G1.4, D7.5), secreted by the selected hybridomas, were isotyped as IgG2a Kappa and were lytic in the presence of complement for 20% lymphocytes from lymph nodes or lung tissue; 40‐50% of lymphocytes from spleen or bone marrow; and 70‐80% of thymocytes or nylon wool nonadherent lung or lymph node lymphocytes. Functional studies showed that prior incubation of either Moab with enriched natural killer (NK) and T lymphocyte subpopulations did not interfere with effector: target conjugate formation but did block the ability of the effector cells to lyse NK‐sensitive targets. We conclude that the Moabs bind to a molecule(s) on NK and T lymphocytes. Further, the preincubation medium acquired the capacity to lyse preferentially NK‐sensitive targets but not NK‐lnsensitive targets. Interestingly, the Moabs were able to induce secretion of lytic factors from thymocytes and to induce resting peripheral T lymphocytes to acquire NK activity and release lytic factors. The ability of the Moab to signal release of lytic factors was retained even when monovalent Fab fragments were used; therefore, the Moabs initiate secretion by mechanisms that do not apparently involve crosslinking or Fc receptor interaction. G1.4 and D7.5 Moabs were functionally and antigenically crossreactive with human, rat, and hamster lymphocytes but not with mouse lymphocytes. Biochemical and immunochemical analysis showed that the G1.4 and D7.5 antigen on thymus cells and lung mononuclear cells has an apparent molecular weight of 27 kD. Although it is unlikely that there is an universal mechanism for secretion used by cells in general, it is interesting that Moab treatment of three different subpopulation of lymphocytes induced secretion of lytic factors and suggests that under physiologic conditions a common molecule might participate in induction of the secretion pathway. These studies lead to the hypotheses that 1) NK and T lymphocytes share a cell surface molecule that participates in signals that initiate secretion and that 2) the expression of the G1.4, D7.5 antigens precedes thymic differentiation.