Katherine Gott

Experimental murine hypersensitivity pneumonitis.

To establish a model of experimental hypersensitivity pneumonitis (EHP) in mice and to examine the influence of genetic background on the pulmonary inflammatory response to Micropolyspora faeni, we determined the responses of C57BL/6, SJL/J, and C3H/HeJ mice to intratracheal (i.t.) injections of M. faeni. Recipient animals received lymph node cells (LNC), peritoneal exudate cells (PEC), and spleen cells (SC) from sensitized mice cultured in vitro with M. faeni. Controls included serum containing anti-M. faeni antibody; uncultured SC from M. faeni-sensitized donors, and M. faeni-cultured SC from ovalbumin (OA)-sensitized donors. Recipients were challenged i.t. with M. faeni or normal saline 48 hr after the cell or serum transfer. We developed a model of EHP in mice. Increasing amounts of i.t. M. faeni were associated with increasing extent of pulmonary inflammation with no difference between the mouse strains. There was substantial increase of the extent of pulmonary abnormalities in the animals receiving cultured SC. The number of transferred cells and the M. faeni concentration correlated with the extent of pulmonary histologic abnormalities. Cultured PEC and LNC could transfer EHP in C3H/HeJ mice only. Serum containing anti-M. faeni antibody, cultured SC from OA-sensitized donors, and noncultured SC from sensitized donors could not transfer EHP. We conclude that it is possible to adoptively transfer EHP.

Th1 Cells That Adoptively Transfer Experimental Hypersensitivity Pneumonitis Are Activated Memory Cells

Abstract. Cultured murine CD4+ T cell lines from Saccharopolyspora rectivirgula–sensitized donors with cytokine secretion characteristics of Th1 cells can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP), whereas Th2 CD4+ cell lines cannot (Cell Immunol 177:169–175, 1997). To assess the differences between these cell lines that may be related to the ability to transfer EHP, we determined cell surface markers that distinguish naive from activated/memory cells that indicate activation and that mediate endothelial adhesion. Both Th1 and Th2 T cell lines are CD4+, CD11a+, ICAM-1+, and L-selectin negative. Th1 cells are CD49d (α4) and LPAM (α4β7) positive, with 32% and 42% of the apparent membrane site density quantitated as the mean molecules of equivalent soluble fluorochromes (MESF) values of unstimulated spleen cells, respectively. Th2 cells are weakly α4 and α4β7 positive, with 15% and 11% of the MESF of unstimulated spleen cells. Th1 cell lines are CD45Rb negative and CD44+, whereas Th2 cell lines are CD45Rb intermediate and CD44−/low. Th1 cells are CD25 (IL-2 receptor) low and Th2 cells CD25 high. We conclude that Th1 cells capable of transfer are activated/memory T cells, and Th2 cells incapable of transfer lack some characteristics of memory/activated T cells (i.e., increase of CD44 and decrease of CD45Rb). Both Th1 and Th2 cell lines express α4β7 and α4 (Th1 > Th2), suggesting that α4 integrin may be important in conferring ability to cells to adoptively transfer EHP.

Experimental hypersensitivity pneumonitis: Role of MCP-1

Abstract Inhalation of Saccharopolyspora rectivirgula causes “farmers lung” disease, a classic example of hypersensitivity pneumonitis (HP). Monocyte chemoattractant protein-1 (MCP-1) is increased in the bronchoalveolar lavage fluid of mice challenged with S rectivirgula, and S rectivirgula induces MCP-1 secretion by alveolar macrophages. We tested the hypothesis that MCP-1 and its receptor CC chemokine receptor-2 (CCR2) are essential to the development of experimental HP by treating mice with MCP-1 antibody and using CCR2−/− mice. Administration of anti–MCP-1 did not change the response to intratracheally administered S rectivirgula. CCR2−/− animals responded in a fashion similar to that of wild-type animals to intratracheally administered.S rectivirgula. To determine the influence of the MCP-1–CCR2 interaction in vitro, we transferred S rectivirgula–cultured spleen cells from S rectivirgula–sensitized mice, to naïve recipients. Later, challenge of the recipients with intratracheal S rectivirgula and examination of both lung histology and bronchoalveolar lavage fluid characteristics were used to determine whether adoptive transfer had occurred. We found that cultured cells from CCR2−/− animals were fully capable of adoptive transfer. We conclude that interaction of MCP-1 with CCR2 is not necessary for the development of pulmonary inflammation in response to intratracheally administered S rectivirgula or cells able to adoptively transfer experimental HP.

Experimental hypersensitivity pneumonitis: influence of Th2 bias

Cultured murine CD4+ cells from Saccharopolyspora rectivirgula sensitized C3H/HeJ (Th1 bias) donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sensitized BALB/c mice (Th2 bias) with S. rectivirgula, obtained spleen and lung associated lymph node (LALN) cells, cultured the cells with specific antigen, and attempted adoptive transfer of EHP. We also treated both C3H/HeJ and BALB/c donor mice with IL4 and anti‐IFNγ before exposure to S. rectivirgula and then cultured cells from both spleen and LALN before attempted transfer of EHP. We found that cultured spleen and lung associated lymph node cells can adoptively transfer EHP in both C3H/HeJ and BALB/c mice as demonstrated by infiltration of the recipient lungs with CD4+ lymphocytes. Treatment of both mouse strains with IL4 and anti‐IFNγ did not change the ability of cultured cells to adoptively transfer EHP. We conclude that EHP induced by S. rectivirgula can occur in animals with either a Th1 or a Th2 bias and is not altered by treatment with IL4 and anti‐IFNγ. This suggests that attributes of the antigen and not genetic background or cytokine environment at the site of initial sensitization determines the results of exposure to S. rectivirgula.

Is IL12 necessary in experimental hypersensitivity pneumonitis

Summary.  Inhalation of Saccharopolyspora rectivirgula (SR) can cause the disease Farmers Lung, a classic example of hypersensitivity pneumonitis. Th1, but not Th2, cell lines can adoptively transfer experimental hypersensitivity pneumonitis (EHP). Substantial amounts of IL12 appear in bronchoalveolar lavage fluid (BALF) after a single intratracheal (IT) injection of SR, and SR‐induced IL12 secretion by both a macrophage cell line and alveolar macrophages. We tested the hypothesis that IL12 is essential for the development of EHP by addition of anti‐IL12 to cultured cells, and adoptive transfer of EHP in IL12p40–/– animals. We transferred SR cultured spleen and lung associated lymph node cells from SR sensitized mice (both IL12p40–/– and wild type), to naïve recipients (both wild type and IL12p40–/–). The addition of anti‐IL12 to cultures of sensitized cells could not ablate the ability of these cells to transfer EHP. Cultured cells from IL12p40–/– animals were fully capable of transferring EHP. In contrast, IL12p40–/– recipients of both wild type and IL12p40–/–‐cultured cells were less able to express EHP (lung histology and BALF characteristics) than wild type mice, and had more eosinophils in both lung tissue and BALF. We conclude that IL12 is not necessary for development of cells able to adoptively transfer EHP, but that it is required for full expression of EHP in recipient animals.

Experimental Hypersensitivity Pneumonitis: Location of Transferring Cells

Abstract. Cultured cells from Micropolyspora faeni-sensitized donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sought to determine the location of transferred cells in recipient animals, the influence of the origin of the cultured cells, and the effect of specific intratracheal challenge. We labeled cultured sensitized spleen or lung-associated lymph node (LALN) cells with CFDA-SE, a cytoplasmic stain, before transfer to naive recipients, which were sacrificed 1 h, 1 day, or 4 days thereafter. We also transferred labeled cultured spleen cells to recipients that were challenged with intratracheal M. faeni and sacrificed 4 days later (MF). Controls were recipients of M. faeni-sensitized and cultured cells challenged with intratracheal normal saline (NS) and recipients of ovalbumin (OVA)-sensitized cells cultured with M. faeni and challenged with intratracheal M. faeni (OVA). The number and proportion of cells that were stained were determined in dispersed spleen, peripheral and lung-associated lymph nodes, and lung parenchyma. The extent of the pulmonary inflammatory response was measured by determining the proportion of microscopic fields that were abnormal and the total number of dispersed pulmonary cells. CFDA-SE stained cells uniformly, and stained cells could be detected in recipients for up to 7 days after transfer. CFDA-SE treatment (0.5 μM) did not affect the ability of cells to transfer EHP adoptively. Transferred cells could be detected easily in lung, lung-associated and peripheral lymph nodes, blood, and spleen. Transferred cells localized to the lung at 1 h but then rapidly decreased with no difference between labeled cells from spleen and LALN. After intratracheal M. faeni challenge, there was no difference in the proportion of labeled cells in the lung among any of the groups (MF, NS, or OVA). There was an increase in the number of lung cells in the MF group compared with the control (NS and OVA) groups. We conclude that cells capable of transfer are transiently (1 h) trapped in the lung but are much decreased in the lung by four days after transfer. After intratracheal antigen challenge of recipients, there is a substantial increase in the number of pulmonary cells in animals exhibiting adoptive EHP but not in the control groups. Transferred cells responsible for EHP are increased in the lungs of animals with adoptive EHP.

EXPERIMENTAL HYPERSENSITIVITY PNEUMONITIS: CELLULAR REQUIREMENTS

We previously demonstrated that Thy1.2+, CD4+, Ia− T cells are responsible for transfer of murine adoptive experimental hypersensitivity pneumonitis (adoptive EHP). To characterize the culture conditions necessary for development of these cells, we depleted cell cultures of Thy1.2+, CD4+, CD8+, or Ia+ cells using MoAbs and complement or magnetic beads, prior to culture of sensitized C3H/HeJ murine spleen cells (SC) with Micropolyspora faeni. After culture, cells were transferred to recipients which were later challenged intratracheally with M. faeni. The extent of pulmonary inflammatory changes in these animals was determined 4 days after intratracheal (i.t.) challenge with M. faeni. Cultured M. faeni‐sensitized SC which had been treated before culture with media, complement only, anti‐CD8 plus complement or magnetic beads alone could transfer EHP to naive animals. SC treated with anti‐Thy1.2 or anti‐CD4 plus complement could not transfer EHP. Treatment of SC with anti‐Iak plus magnetic beads diminished the ability of cultured cells to transfer EHP. We conclude that the ability to produce cells able to adoptively transfer EHP is dependent on the presence of Thy1.2+, CD4+ and Ia+ cells, but not CD8+ cells, at the onset of culture.

Mast cell hyperplasia in experimental hypersensitivity pneumonitis

We investigated the presence of mast cells in a model of experimental hypersensitivity pneumonitis (EPH). Guinea pigs exposed to 8 weekly intratracheal challenges with Micropolyspora faeni exhibited significant increases in the number of mast cells within the lung as compared to controls and animals challenged only 2 or 4 times. The number of cells in M. faeni-challenged animals were increased around bronchi, bronchioles, blood vessels and in alveolar septa. There appeared to be contraction of peribronchial, peribronchiolar and vascular smooth muscle. Ultrastructural examination of lung tissue revealed the presence of degranulating mast cells. Bronchoalveolar lavage histamine levels were increased after 8 but not after 2 or 4 weekly challenges. Serum anti-M. faeni antibody was present in all M. faeni-exposed animals but not in control animals. We conclude that mast cells and histamine levels in bronchoalveolar lavage fluid are increased in a model of EHP caused by repetitive, intratracheal injection of M. faeni particulate antigen.