Lymphocyte migration into different lung compartments during an antigen induced inflammation: Is the spleen a major reservoir of these lymphocytes?

The hypothesis was tested whether lymphocytes of immunized and pulmonary challenged LEW rats adhere in higher numbers to the lung vascular bed than control lymphocytes and whether these immigrating cells come from the spleen. The kinetic of a primary immune response to sheep red blood cells (SRBC) was characterized in different lung compartments such as the vascular marginal pool, the interstitium and the bronchoalveolar space. The adherence of genetically labeled splenocytes from SRBC-immunized and challenged rats and from non-challenged rats was investigated in challenged lungs using the ex vivo system of the isolated buffer-perfused lung (IPL). Furthermore, immunized animals were splenectomized and challenged with SRBC. It was found that lymphocytes were increased with a maximum in the lung interstitium on day 3 and in the bronchoalveolar lavage fluid (BALF) on day 4. The adhesion to the pulmonary vascular endothelium of splenic T cells from SRBC-immunized rats in the IPL was not significantly increased compared to those from control animals. A significant transmigration from the vasculature into the BALF was not found. On day 4 after challenge the cell numbers in the lung compartments of the splenectomized animals were comparable to controls. The spleen alone has no significant role as a source of lymphocytes in lung inflammation. Therefore, the pulmonary immune response seems to be triggered mainly by the local environment and not by the accompanying systemic immune reaction.     

Recruitment of lymphocytes and dendritic cells from the blood to the bronchoalveolar space and the draining lymph nodes after a single intrabronchial application of the lipopeptide MALP-2.

OBJECTIVE: It has been shown previously that the synthetic macrophage-activating lipopeptide, MALP-2, is a potent stimulator of the respiratory immune system and an effective adjuvant in the induction of mucosal immune responses. In this study, the migration route of leukocytes from the blood to the bronchoalveolar space and then to the draining lymph nodes was investigated. METHODS: MALP-2 was intratracheally instilled into lungs of Lewis rats. Bronchoalveolar lavage cells as well as cell preparations of other lung compartments such as the marginal vascular pool, the interstitial pool and also the draining lymph nodes were examined 3 days later. RESULTS: The application of MALP-2 induced a pronounced leukocyte accumulation in the bronchoalveolar space and the lung interstitium but not in the marginal vascular pool. A tendency to increased lymphocyte and dendritic cell numbers was observed in the draining lymph nodes. CONCLUSION: Our data indicate the migration of blood cells into the lung interstitium and the bronchoalveolar space in response to MALP-2. Thus, the immune reaction induced by MALP-2 might be of relevance as an adjuvant treatment in inhalant vaccination strategies in the lung.     

Lung Compartmentalization of Inflammatory Cells in Sepsis

Lung injury commonly occurs in the setting of systemic inflammatory response syndrome occurring during bacterial sepsis. There has been little work quantifying different leukocytes within the different compartments of the lung and their association with overt lung injury in sepsis. We examined the pathogenesis of lung injury after cecal ligation and puncture (CLP), a clinically relevant model of sepsis. To assess the sequestration and migration of leukocytes, leukocyte differentials were obtained for the lung vascular compartment and the bronchoalveolar airspace. At 24 h post CLP, there were signs of edema in the lung, while at 48 h after CLP, there were clear indications of alveolar wall thickening with increased cellularity and diffuse alveolar hemorrhage. The number of lymphocytes in the pulmonary vascular compartment dropped by 50% and doubled in the (bronchoalveolar lavage) BAL, 24 h after CLP compared to sham controls suggesting that there was transendothelial migration of lymphocytes. At 48 h after CLP, lymphocyte numbers in the vasculature was similar to controls but BAL lymphocyte numbers were still raised. The number of pulmonary intravascular neutrophils were similar to controls at 24 h post CLP but were greatly elevated 48 h after CLP. The increase in neutrophils was partly due to a substantial increase in the percentage of immature band cells, indicating recruitment of neutrophils from the bone marrow. There were very few neutrophils in the BAL of sham controls and CLP rats. Perfusate monocyte/macrophages were significantly increased 48 h after CLP and a similar increase in macrophages was observed in the BAL. These results strongly suggest a role for lymphocytes and macrophages in the development of overt lung injury as the migration of these cells corresponds to that of the appearance of lung injury 48 h after CLP. Importantly our data also demonstrates the compartmentalization and migration of different inflammatory cell-types during the development of sepsis.     

Resolution of allergic airways inflammation but persistence of airway smooth muscle proliferation after repeated allergen exposures

BACKGROUND: Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. OBJECTIVE: We investigated the long-term effects of repeated allergen exposure. METHODS: Brown-Norway (BN) rats sensitized to ovalbumin (OVA) were exposed to OVA or saline aerosol every third day on six occasions and studied 24 h, 7 days and 35 days after the final exposure. We measured airway inflammation, ASM cell proliferation (by incorporation of bromodeoxyuridine; BrdU) and bronchial responsiveness to acetylcholine. RESULTS: At 24 h, in OVA-exposed rats, we detected elevated OVA-specific serum IgE, increased numbers of macrophages, eosinophils, lymphocytes and neutrophils in the bronchoalveolar lavage (BAL) fluid and increased numbers of MBP+ (major basic protein) eosinophils and CD2+ T cells within the bronchial submucosa. This coincided with increased numbers of ASM cells expressing BrdU and with bronchial hyper-responsiveness (BHR). At 7 days, BHR was detected in OVA-exposed rats, coincident with increased numbers of macrophages and lymphocytes in BAL fluid together with increased numbers of CD2+ T cells within the bronchial submucosa. This coincided with increased numbers of ASM cells expressing BrdU. By day 35, the number of ASM cells expressing BrdU remained elevated in the absence of cellular infiltration and BHR. CONCLUSION: Repeated OVA-challenge results in persistent ASM cell proliferation in the absence of bronchial inflammation and BHR, which lasts for at least 1 week following cessation of exposure.     

Absolute numbers of lymphocyte subsets migrating through the compartments of the normal and transplanted rat spleen

Lymphocyte migration is one of the basic principles of the immune system. Up to now lymphocyte migration experiments have been performed either in a quantitative way, determining whole organ recoveries of radiolabeled lymphocytes without histologic localization, or based on autoradiography which does not provide absolute numbers of immigrant lymphocytes. In this study the traffic of lymphocyte subsets through the splenic compartments: red pulp (RP), marginal zone (MZ), periarteriolar lymphatic sheath (PALS) and follicle was evaluated in absolute numbers. In normal spleens and splenic transplants fluorescein isothiocyanate (FITC)-labeled immigrant lymphocytes were localized and characterized immunohistochemically in cryostat sections by light microscopy. In addition morphometry of the splenic compartments was performed and the recovery of ⁵¹Cr-labeled lymphocytes in the spleen was determined. The combination of these methods allowed total numbers of immigrant subset cells to be calculated in individual splenic compartments. At 15 min about 17% of the injected B lymphocytes were found in the MZ. This is the largest fraction of an injected lymphocyte subset found in a single splenic compartment. At 24 h immigrant B cells were not only found in the follicle, but they had reached comparable numbers in the three compartments: follicle, RP and MZ. Most immigrant T lymphocytes were found in the PALS, which from 6 h after injection onwards contained more T cell immigrants than any single organ of the body. CD4⁺ and CD8⁺ lymphocytes showed a similar distribution throughout the splenic compartments at early time points. At 24 h CD4⁺ lymphocytes homed preferentially to the PALS, whereas CD8⁺ cells seemed to prefer the RP and MZ. Both CD4⁺ and CD8⁺ cells also migrated into the follicles. In regenerated splenic tissue after autotransplantation lymphocyte immigration was reduced in all compartments and to the MZ in particular. An impaired lymphocyte migration to the MZ in splenic transplants may be one reason for the lack of protection provided against bacterial infections. Thus examining lymphocyte migration in absolute numbers provides additional information which cannot be gained by determining labeling indices or percentages of lymphocyte subsets alone.     

Lymphocyte subsets in distinct lung compartments show a different ability to produce interferon-gamma (IFN-γ) during a pulmonary immune response

Lymphocytes play an important immunoregulatory role in pulmonary immune responses. By releasing cytokines they can control the cell–cell communication of other participating cells. Although it is well established that the lung lymphocytes, localized in distinct compartments, differ in their subset composition, little is known about cytokine production in these compartments during immune responses. Lewis rats were immunized by intravenous administration of sheep erythrocytes on day 0 and day 7 and challenged intratracheally with sheep erythrocytes on day 10. Four days after intratracheal (i.t.) challenge the composition of lymphocyte subsets (CD2⁺, CD4⁺, CD8⁺, B cells, natural killer (NK) cells) in the spleen, blood, lung perfusate, lung tissue and bronchoalveolar lavage fluid (BALF) was characterized, and intracellular IFN-γ was detected in these subsets by flow cytometry. Comparing control and immunized animals, no changes were found in lymphocyte numbers, subsets or the percentage of IFN-γ-producing lymphocytes in the spleen, blood and lung perfusate. In lung tissue and BALF, however, the absolute number of all lymphocyte subsets and the percentage of IFN-γ-producing lymphocytes were increased. When the lymphocyte subsets were analysed an increased percentage of IFN-γ-producing T cells was found in lung tissue (4.5 ± 0.6% versus 12.8 ± 1.1%) and in BALF (7.8 ± 1.4% versus 14.8 ± 1.9%) of immunized animals opposed to controls, this increase being seen in both CD4⁺ and CD8⁺ cells. Thus, there is an accumulation of T cells with an increased potential to produce IFN-γ in the lung interstitium and the bronchoalveolar space during pulmonary immune responses.     

The role of lymphocyte production and migration in the lymphopenia caused by 2-acetyl-4-tetrahydroxybutyl imidazole.

2-Acetyl-4-tetrahydroxybutyl imidazole (THI), a component of the food colouring ammonia caramel, has been shown to produce a profound and rapid lymphopenia in peripheral blood in the rat. In order to investigate whether the cause of the lymphopenia was due to the reduced production and influx in the circulation, redistribution of lymphocytes into other lymphoid compartments or an increased cell death, THI (1 mg/kg/day) was given in the drinking water for up to 14 days to F344 rats. A profound depletion of lymphocytes after already 1 day was only found in the blood compartment, whereas no such marked and rapid changes were found in the cellularity of other lymphoid compartments. The proportion and absolute number of DNA-synthesizing cells in each lymphoid organ was quantified using an antibody directed against incorporated 5-bromo-2'-deoxyuridine (BrdU), 1 h after a single BrdU injection. Additionally, enumeration and localization of BrdU+ cells was determined at later time points after a single BrdU injection by flow cytometry and immunocytochemistry, in order to examine the distribution and localization of recently formed (BrdU+) lymphocytes. THI treatment had no effect on the proliferation rate and the distribution of newly formed (BrdU+) cells in the lymphoid organs. However, migration studies revealed that THI treatment resulted in an increased percentage of fluorescein-labelled peripheral blood lymphocytes found in the spleen and bone marrow and a decreased percentage in the cervical and mesenteric lymph nodes, 24 h after injection. Collectively these results indicate that the lymphopenia in the peripheral blood compartment after THI treatment, is caused by a rapid sequestration of lymphocytes into the spleen and bone marrow rather than by a reduced lymphocyte production and release into the periphery. The fact that THI also caused lymphopenia in splenectomized rats, indicates that the spleen does not play an active part in the change in migrational behaviour of lymphocytes after THI treatment. Finally, as there was no increase in the absolute number of lymphocytes found in the spleen or bone marrow it seems they are rapidly degraded.     

Bronchoalveolar lavage cell analysis and lung function impairment in patients with systemic lupus erythematosus (SLE).

We examined the relationship between peripheral blood and bronchoalveolar lavage (BAL) lymphocyte phenotypes and lung function in 19 patients with SLE, and evaluated their association with disease activity. Lung function assessment showed a mildly restrictive pattern with frequent impairment of transfer factor for carbon monoxide (T1,co) and diffusing capacity of the alveolocapillary membrane (Dm), of late-expiratory airflow rates and with a high prevalence of increased airway resistance. T1,co, Kco and Dm correlated inversely with the numbers of CD8+ cells and CD56+/CD16+/CD3- (NK) cells in BAL. Oxygen radical production, both by stimulated and unstimulated BAL cells and blood polymorphonuclear leucocytes (PMN) was significantly increased in SLE. In comparison with healthy controls, patients with SLE had a lower percentage of CD19+ B cells in the BAL versus an increased percentage of these cells in peripheral blood. HLA-DR expression on CD4+ and CD8+ lung lymphocytes was markedly increased in SLE. Current SLE disease activity was not associated with changes in BAL or peripheral blood lymphocyte phenotypes. Our data suggest that an ongoing cell-mediated immune response is present in the lungs in SLE, particularly involving activated CD8+ T cells and CD56+/CD16+/CD3- NK cells. It is associated with up-regulated local production of oxygen radicals and with impaired pulmonary diffusing capacity. This inflammatory process seems to be independent of general SLE disease activity.     

The Marginal Blood Pool of the Rat Contains not only Granulocytes, but also Lymphocytes, NK-Cells and Monocytes: a Second Intravascular Compartment, its Cellular Composition, Adhesion Molecule Expression and Interaction with the Peripheral Blood Pool

To leave the blood, leucocytes marginate to the vessel wall. Granulocytes thereby form the so-called marginal pool. It is unclear to what extent such a second intravascular compartment also exists for lymphocyte subsets, NK-cells and monocytes. Samples of the peripheral blood and the marginal pool of the LEW rat were analysed by flow cytometry. In the marginal pool the percentage of granulocytes and monocytes was significantly higher compared to that of the peripheral blood, and the proportion of 'naive' T and B lymphocytes was decreased. The expression of LFA-1 was higher on all leucocyte subsets of the marginal pool except the granulocytes, whereas no differences were seen for the expression of other adhesion molecules (α₄-integrins, ICAM-1, CD2, L-selectin, and CD44). In addition, splenectomy influenced the cellular composition of peripheral blood and marginal pool differently and, after injection of blood leucocytes, these cells were found in both compartments showing its characteristic cellular composition. Thus, not only granulocytes, but also B and T lymphocyte subsets, NK-cells and monocytes form a second distinct intravascular compartment. This marginal pool probably influences the cellular composition of leucocyte subsets available for entry into the tissues.     

Quantification of proliferating lymphocyte subsets appearing in the intestinal lymph and the blood.

Lymphocyte emigration from the intestinal wall via lymphatics is necessary to maintain gastrointestinal immunity and also to connect the different parts of the mucosal immune system. In the present study the numbers and time kinetics of proliferating lymphocyte subsets leaving the gut wall via intestinal lymphatics were analysed in mesenteric lymph node adenectomized minipigs (n = 8). After cannulation of the major intestinal lymph duct, afferent lymph was collected under non-restraining conditions. In four pigs lymphocytes taken from the intestinal lymph and blood were incubated in vitro with the thymidine analogue bromodesoxyuridine (BrdU) to label all lymphocytes in the S-phase of the cell cycle. The other four pigs received a single i.v. injection of BrdU 1 week after cannulation. The initial percentage of BrdU+ lymphocyte subsets in the intestinal lymph 15 min after BrdU injection was comparable to that after the in vitro labelling (1.5 +/- 0.7% in T cells, 10.6 +/- 1.6% in IgM+ cells and 30.0 +/- 11.9% in IgA+ cells). From this level onwards, the percentage of in vivo labelled BrdU+ lymphocyte subsets reached a maximum at 12 h after BrdU application. A different pattern of BrdU+ subsets was seen in the blood. After an early peak at around 3-4 h, the frequency of BrdU in vivo labelled cells decreased. Each subset had a maximum between 12 h and 48 h after BrdU application (maximum of BrdU+ CD2+ T cells at 12 h, 4.6 +/- 1.5%; IgM+ BrdU+ at 48 h, 8.8 +/- 3.3%). The present results provide a basis to determine the time necessary for induction of specific intestinal immunity during oral vaccination studies.     

Recruitment of antigen-specific Th1-like responses to the human lung following bronchoscopic segmental challenge with purified protein derivative of Mycobacterium tuberculosis

Although the mechanisms of specific immunity to Mycobacterium tuberculosis in humans are poorly understood, responses of Th1-like CD4+ T cells appear to be essential for protection. We hypothesized that healthy individuals displaying positive skin-test responses to purified protein derivative of M. tuberculosis (PPD) would have the capacity to mobilize M. tuberculosis-specific Th1 cells to the lung in response to bronchoscopic challenge with PPD. Local instillation of 0.5 tuberculin units of PPD was followed 48 h subsequently by bronchoalveolar lavage (BAL) of PPD-challenged and control segments. In PPD-positive subjects, PPD challenge resulted in a 2.7-fold increase in total BAL cells and in an increase in the percentage of lymphocytes in BAL from 10 to 19%. The BAL lymphocytosis observed in PPD-challenged segments was characterized by an increased percentage of CD4+ T cells and by increased numbers of cells capable of antigen-specific interferon-gamma production. In contrast, PPD-negative subjects did not develop local inflammation following PPD challenge. These findings indicate that bronchoscopic challenge with PPD results in recruitment of antigen-specific recall responses to the lung. This novel approach may be useful in clarifying the basis of local immunity against M. tuberculosis, and could serve more generally as a model of the development of Th1-like responses in the human lung.     

The Influence of Age and Breeding Conditions on the Number and Proliferation of Intraepithelial Lymphocytes in Pigs

The number and subset composition of intraepithelial lymphocytes (IEL) were studied in pigs in different age groups ranging from 1 day to 4.5 years. There were no major differences between the numbers of IEL in the jejunum and ileum. The postnatal increase of IEL largely depended on the breeding conditions: in germ-free animals there was a constant level, while in conventionally bred pigs the numbers increased more obviously than in specified pathogen-free (SPF) pigs. As the numbers of IEL can rise due to increased influx from other organs, decreased emigration, decreased apoptosis or local proliferation, the incorporation of the DNA precursor bromodeoxyuridine (BrdU) by IEL was studied after different labelling protocols. There were low but definite numbers of BrdU+ IEL 1 h after BrdU, indicating in-situ proliferation in conventional animals with a tendency to a higher index in the jejunum than the ileum. Repetitive labelling protocols for 14 days resulted in 12-20% BrdU+ IEL, which may be caused by local proliferation and immigration of lymphocytes produced in other lymphoid organs as documented for the pig. Future studies need to focus on the factors regulating local proliferation and the migration of IEL in different species.     

Selective attrition of non-recirculating T cells during normal passage through the lung vascular bed.

Transient arrest of T lymphocytes in the lung vascular bed following infusion of cells subjected to in vitro manipulations has been recognized for many years as a troublesome 'artefact', and has generally been attributed to trauma-induced changes in lymphocyte surface membranes. However, a number of laboratories have reported that the trapping process also occurs under situations where lymphocyte surface damage is minimal or absent, suggesting that the phenomenon may represent an intrinsic component of normal lymphocyte circulation. Consistent with these suggestions, recent studies from our laboratory have demonstrated the presence of large numbers of T cells in collagenase digests of normal peripheral lung tissue, which cannot be removed by broncho-alveolar lavage or perfusion of the tissue vascular bed. In the present experiments we have characterized these lung T cells in SPF rats. The properties common to this population include hydroxyurea sensitivity, high CD8:CD4 ratio and high frequency of cells recently derived from the thymus, and saturation thymidine-labelling studies indicated that greater than 90% of the lung T cells had divided within a 14-day test period. Additionally, cloning studies under conditions of limiting dilution indicate markedly reduced capacity for proliferation, relative to T cells in blood or spleen. We interpret these data to indicate that selective trapping and subsequent down-regulation of non-recirculating T cells is a normal consequence of passage through the lung vascular bed.     

Phenotypic changes to the endogenous antigen-specific CD8+ T cell response correlates with the development and resolution of allergic airway disease

The role of CD8(+) T cells in the pathogenesis of asthma remains controversial, as both pro- and anti-inflammatory functions have been suggested. This study was designed to examine the endogenous CD8(+) T cell response in a biphasic ovalbumin (OVA)-induced model of allergic airway disease (AAD) and its subsequent resolution with the development of local inhalational tolerance (LIT). We observed increases in OVA-specific CD8(+) T cell numbers in the local lung compartments (bronchoalveolar lavage, lung tissue, hilar lymph node) at AAD and LIT; systemic compartments (spleen, inguinal lymph node) displayed no such increases in CD8(+) T cell numbers. OVA-specific CD8(+) T cells appeared to exhibit plasticity both phenotypically and functionally. They possessed pro-inflammatory characteristics at AAD, with high phenotypic expression of CD11a and increased functional expression of granzyme B and interferon-γ. In contrast, at LIT they showed increased phenotypic expression of the inhibitory marker NKG2A and functionally did not produce granzyme B or interferon-γ. In addition, in a discontinuous model the OVA-specific CD8(+) T cells could be recalled on re-exposure to OVA, demonstrating memory. Finally, confocal microscopy results showed that OVA-specific CD8(+) T cells at AAD are associated with B cell aggregates in lung tissue. These B cell aggregates resembled tertiary ectopic lymphoid tissue and may thus provide a local environment for the salient cellular interactions that contribute to the development of LIT.     

Accessory cell function of human alveolar macrophages in antigen-induced T lymphocyte proliferation

We compared the accessory cell function of human alveolar macrophages (AM) to that of human blood monocytes (Mo) obtained by bronchoalveolar lavage and venipuncture from normal volunteers. Graded numbers of either AM or Mo were added to autologous peripheral blood T lymphocytes that were stimulated with a purified protein derivative of tuberculin (PPD). Either AM or Mo were cocultured with allogeneic T lymphocytes in mixed lymphocyte reaction (MLR) experiments. Both AM and Mo supported the PPD-induced T lymphocyte proliferation and allogeneic MLR at low ratios of AM or Mo to T lymphocytes with similar efficiency. However, AM showed marked suppressive effects at higher ratios of AM to T lymphocytes (1:1). PPD-pulsed AM, but not AM killed by physical treatments (heat, freeze-thaw, sonication), induced T lymphocyte proliferation. An indirect immunofluorescent study demonstrated that most AM express HLA-DR antigens. Furthermore, AM synthesized DR antigens with molecular weights of 33,000 and 29,000-31,000 daltons. When AM were treated with both anti-DR monoclonal antibody and complement, PPD-induced T lymphocyte proliferation and MLR were diminished. These results suggest that human AM function as accessory cells in the antigen-induced T lymphocyte proliferation and DR antigens on AM play an important role in the accessory cell function.     

Cellular changes in the bronchoalveolar lavage (BAL) of pigs, following immunization by the enteral or respiratory route.

Normal young pigs were immunized by the oral or aerogenic route with the viable or inactivated lung-pathogenic bacterium Actinobacillus (Haemophilus) pleuropneumoniae. Three weeks later the cellular composition as well as the lymphocyte subset composition of the bronchoalveolar space were examined by BAL. Lymphocytes in the lavage increased significantly, including CD4+ and CD8+ T cells. After oral immunization a dramatic increase of plasma cells and lymphoid blasts was found. Among immunoglobulin-positive lymphocytes IgG+ cells showed the most pronounced increase. For most lymphocyte subsets there was no difference between viable and inactivated bacteria. Oral immunization with a lung-pathogenic bacterium results in increased numbers of lymphocytes in the bronchoalveolar space and might play a critical role in protection against lower respiratory tract infections.     

Lymphocytes in the lung: an often neglected cell

The lung is continuously in contact with inhaled particles, some of which are of microbial origin. This requires adequate defence mechanisms in the form of immune reactions. These can be subdivided into the afferent and efferent limb. Specific immune reactions depend on the interactions between lymphoid and accessory cells. Therefore, the local histotopographic localization of lymphocyte subsets has to be known to understand pulmonary immune reactions. As lymphocytes have often not been mentioned when cells in the respiratory tract have been characterized, their compartmentalization, number and subset composition in the lung are outlined here. Lymphocytes are found in the epithelium and lamina propria of the bronchi with different subset compositions. In some species, like the rabbit, bronchus-associated lymphoid tissue (BALT) is found as follicle-like aggregations with lymphocytes infiltrating the epithelium, which shows specialized epithelial cells. BALT, however, is not a constitutive structure in all species, e.g in humans. Nevertheless, certain (probably) microbial stimuli can induce BALT in adult humans. In contrast to many other organs, the lung vascular bed contains large numbers of lymphocytes. Little is known about the adhesion molecules that make this margination possible. In the lung interstitium about 10×10⁹ lymphocytes have been calculated for healthy adults. The most easily accessible pool of lymphocytes in the human lung are those recovered by bronchoalveolar lavage. The vast majority of such lymphocytes express markers typical for memory lymphocytes. The intrapulmonary migratory routes of lymphocytes and the integration of the lung in the common mucosal immune system are described. A multicompartmental model for lymphocytes is outlined as a basis for understanding lung diseases such as asthma.     

Proliferating macrophages, dendritic cells, natural killer cells, T and B lymphocytes in the middle ear and Eustachian tube mucosa during experimental acute otitis media in the rat.

Although many studies focus on the increase of immunocompetent cells within the middle ear mucosa during acute otitis media it is poorly understood how this increase is mediated. The differentiation between two possible causes, i.e. immigration and local proliferation, would help to better understand the pathophysiology of this disease. Therefore, the number of proliferating macrophages, dendritic cells, natural killer cells and T and B lymphocytes was studied during acute otitis media in the rat middle ear mucosa (ME mucosa) and Eustachian tube mucosa (ET mucosa) by labelling proliferating leucocytes with the DNA precursor bromodeoxyuridine (BrdU). By removing the middle ear and Eustachian tube 24 h after BrdU injection, the contribution of immigrated newly formed cells was estimated. At this timepoint, many leucocytes in the ME and ET mucosa had incorporated BrdU (between 15 and 25% within the subsets). By analysing these tissues one hour after BrdU injection, the local proliferation rate was determined (between 2 and 9% within the subsets). Thus, the inflamed ME and ET mucosa are the destination of immunocompetent cells and, as our data show, the inflamed microenvironment supports local proliferation of immunocompetent cells.     

In vivo effects of a synthetic 2-kilodalton macrophage-activating lipopeptide of Mycoplasma fermentans after pulmonary application

Mycoplasmas can cause interstitial pneumonias inducing critical illness in humans and animals. Mycoplasma infections are characterized by an influx of neutrophils, followed by an accumulation of macrophages and lymphocytes. The present study deals with the question of which mycoplasmal components cause this host reaction. The mycoplasma-derived, macrophage-activating lipopeptide 2S-MALP-2 was used to mimic the sequelae of a mycoplasma infection. To this end, 2S-MALP-2 was intratracheally instilled into the lungs of Lewis rats, and the bronchoalveolar lavage cells were examined at different times after different doses of 2S-MALP-2. Application of 2.5 microg induced a pronounced leukocyte accumulation in the bronchoalveolar space. At 24 h after 2S-MALP-2 administration, the majority of leukocytes consisted of neutrophils, followed by macrophages, peaking on days 2 and 3. Lymphocyte numbers, although amounting to only a few percent of the total bronchoalveolar lavage cells, also increased significantly, with maximal lymphocyte accumulation occurring by 72 h after instillation. The leukocyte count of the lung interstitium was increased on day 3 after treatment. After 10 days all investigated cell populations returned to control levels. Transient chemotactic activity for neutrophils was detected in the bronchoalveolar lavage fluid early after 2S-MALP-2 application, followed by monocyte chemoattractant protein-1 activity (MCP-1) in lung homogenates. MCP-1 was produced by bronchoalveolar lavage cells upon stimulation with 2S-MALP-2. Our data indicate that mycoplasmal lipoproteins and lipopeptides are probably the most relevant mycoplasmal components for the early host reaction. The primary target cells are likely to be the alveolar macrophages liberating chemokines, which attract further leukocytes.