Microbial Pathogenesis 1991 ; 11 : 149-157
Sendai virus infection of normal and protein malnourished mice : response of airway leukocytes to infection Hoan Jong Lee,' Curtis T . Moody,' Carol S . Reiss, 3 Victor Pena-Cruz' and Kenneth McIntosh'* 'Division of Infectious Diseases, and 'Division of Immunology, The Children's Hospital, and 'Division of Pediatric Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, U.S.A . (Received October 27, 1990 ; accepted April 15, 1991)
Lee, H . J . (Division of Infectious Diseases, Dana Farber Cancer Institute, Boston, Massachusetts, U .S .A .), C . T . Moody, C. S . Reiss, V . Pena-Cruz and K . McIntosh . Sendai virus infection of normal and protein malnourished mice : response of airway leukocytes to infection . Microbial Pathogenesis 1991 ; 11 : 149-157 . We examined the cells recovered by bronchoalveolar lavage (BAL) during the course of respiratory infection caused by Sendai virus in normal (20% protein diet) and malnourished (2% protein diet) mice . As in our previous experiments, mortality in normal mice was 32% in comparison with 87% in the malnourished group . Virus was isolated until the 5th day in normally fed mice and until the 9th day in the malnourished group . BAL fluids contained 97% macrophages before infection in both groups . During infection there was a progressive lymphocyte response, reaching a peak of 60-70% on day 5 in the normal mice and on days 7-9 in the malnourished group . Subtyping of BAL cells, by flow cytometry indicated that in uninfected animals lymphocytes were largely CD4-bearing . On days 3 and 5 post-infection most mononuclear cells were Thy 1 .2-positive, but lacked both CD4 and CD8 markers and were therefore probably natural killer cells . Beginning on day 5, in both diet groups CD8-positive cells rose to become the predominant subset . In the 20% protein diet group, CD8-positive cells reached a maximum of 60% on day 7, whereas in the 2% protein diet group this level was not reached until day 9 . These results were consistent in three separate experiments . In malnourished mice the delayed appearance of CD8-bearing cells in the airway may contribute to the higher mortality and delayed virus clearance during Sendai virus infection . Key words : Sendai virus ; pneumonia ; malnutrition ; respiratory immunity ; lymphocyte .
Introduction Acute respiratory infection is an important cause of morbidity and mortality in children under 5 years of age in most of the developing world .' Malnutrition is thought to play a significant role in this public health problem . 2 ' The mechanism is, however, poorly understood . It is widely assumed that the major reason for the high mortality is an increased incidence of severe bacterial pneumonia .' On the other hand, viral lower respiratory infections are also frequent in children residing throughout the Third World s
* Author to whom correspondence should be addressed at : 300 Longwood Avenue, Boston, MA 02115, U .S .A . 0882-4010/91/090149+09 S03 .00/0
© 1991 Academic Press Limited
H . J . Lee et al.
1 50
and there is little or no information about the severity of pure viral respiratory infection in this group . We have previously reported that the mice with protein malnutrition demonstrated increased susceptibility to, and greater morbidity and mortality after intranasal Sendai virus infection .' Sendai virus is a natural murine pathogen and is widely used in animal experiments as a model of respiratory viral infection .' It produces an interstitial pneumonia which is histologically similar to viral pneumonia and bronchiolitis in children . Furthermore, the virus biochemically and physically resembles parainfluenza virus type 1, a major pediatric pathogen, and is in the same family as respiratory syncytial virus . Recent publications suggest that flow cytometric analysis of bronchoalveolar lavage (BAL) cells may provide a simple method of quantitating the airway component of the immune response after respiratory infection ."" We decided, therefore, to analyse the sequential changes in the cells recovered by BAL in order to investigate the pathogenetic mechanisms involved in the increased susceptibility to Sendai virus infection in malnourished mice . Results We repeated the central experiments three times with almost identical results . Because there were minor variations between experiments in the ages of the mice (3 weeks versus 5 weeks) and doses of virus [1 x 103 plaque forming units (pfu) versus 2 x 10 3 pfu], we describe mainly the results of a single experiment performed on 3-week-old mice with a 1 x 103 pfu inoculum . As in previous experiments,' mice on the 2% protein diet failed to gain weight during the course of the 2-week preparatory period . At the time of inoculation, proteindeprived mice were entirely normal in appearance and behavior but weighed, on average, 55% of the normally fed mice . Most mortality occurred between days 5 and 9, and exaggerated mortality in the 2% group was observed between days 5 and 8 . As in our previously published work, the overall mortality of the 2% diet group was 87% by the Kaplan-Meier method, whereas that of the 20% protein diet group was 32% (Fig . 1) . We also found, as in previous
100 80 0 60 5
N
40 20 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time after viral inoculation (days)
Fig . 1 . Survival rate of BALB/c mice after intranasal inoculation of Sendai virus, 1 x 103 pfu per animal (Kaplan-Meier method) . Three to five animals were killed every other day for analysis of bronchoalveolar lavage fluid cells . In this experiment 51 mice fed a 20% protein diet for two weeks before inoculation weighed 17 .8±1 .2 g (mean±SD) on day 0 ; 49 were inoculated for the experiment . Seventy-eight mice fed a 2% protein diet for the same interval weighed 9 .8±1 .2 g on day 0 ; all 78 were inoculated for the experiment . 0 : 20% protein diet; 0 : 2% protein diet .
Sendai virus in malnourished mice
1 51
Fig . 2 . Pulmonary virus titer in BALB/c mice with intranasal Sendai virus infection . The lungs were homogenized after bronchoalveolar lavage for analysis of cells . Each point represents mean±SD of three to five mice . The circle at day 9 (2% protein diet group) represents the titer from a single mouse (of five sacrificed in that group) in which virus was grown . Titers are plotted as loglo pfu/g of mouse lung . o : 20% protein diet ; 0 : 2% protein diet .
experiments, that pulmonary virus titers were significantly higher and lasted longer in the malnourished mice (P
Sendai virus infection of normal and protein malnourished mice : response of airway leukocytes to infection Hoan Jong Lee,' Curtis T . Moody,' Carol S . Reiss, 3 Victor Pena-Cruz' and Kenneth McIntosh'* 'Division of Infectious Diseases, and 'Division of Immunology, The Children's Hospital, and 'Division of Pediatric Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, U.S.A . (Received October 27, 1990 ; accepted April 15, 1991)
Lee, H . J . (Division of Infectious Diseases, Dana Farber Cancer Institute, Boston, Massachusetts, U .S .A .), C . T . Moody, C. S . Reiss, V . Pena-Cruz and K . McIntosh . Sendai virus infection of normal and protein malnourished mice : response of airway leukocytes to infection . Microbial Pathogenesis 1991 ; 11 : 149-157 . We examined the cells recovered by bronchoalveolar lavage (BAL) during the course of respiratory infection caused by Sendai virus in normal (20% protein diet) and malnourished (2% protein diet) mice . As in our previous experiments, mortality in normal mice was 32% in comparison with 87% in the malnourished group . Virus was isolated until the 5th day in normally fed mice and until the 9th day in the malnourished group . BAL fluids contained 97% macrophages before infection in both groups . During infection there was a progressive lymphocyte response, reaching a peak of 60-70% on day 5 in the normal mice and on days 7-9 in the malnourished group . Subtyping of BAL cells, by flow cytometry indicated that in uninfected animals lymphocytes were largely CD4-bearing . On days 3 and 5 post-infection most mononuclear cells were Thy 1 .2-positive, but lacked both CD4 and CD8 markers and were therefore probably natural killer cells . Beginning on day 5, in both diet groups CD8-positive cells rose to become the predominant subset . In the 20% protein diet group, CD8-positive cells reached a maximum of 60% on day 7, whereas in the 2% protein diet group this level was not reached until day 9 . These results were consistent in three separate experiments . In malnourished mice the delayed appearance of CD8-bearing cells in the airway may contribute to the higher mortality and delayed virus clearance during Sendai virus infection . Key words : Sendai virus ; pneumonia ; malnutrition ; respiratory immunity ; lymphocyte .
Introduction Acute respiratory infection is an important cause of morbidity and mortality in children under 5 years of age in most of the developing world .' Malnutrition is thought to play a significant role in this public health problem . 2 ' The mechanism is, however, poorly understood . It is widely assumed that the major reason for the high mortality is an increased incidence of severe bacterial pneumonia .' On the other hand, viral lower respiratory infections are also frequent in children residing throughout the Third World s
* Author to whom correspondence should be addressed at : 300 Longwood Avenue, Boston, MA 02115, U .S .A . 0882-4010/91/090149+09 S03 .00/0
© 1991 Academic Press Limited
H . J . Lee et al.
1 50
and there is little or no information about the severity of pure viral respiratory infection in this group . We have previously reported that the mice with protein malnutrition demonstrated increased susceptibility to, and greater morbidity and mortality after intranasal Sendai virus infection .' Sendai virus is a natural murine pathogen and is widely used in animal experiments as a model of respiratory viral infection .' It produces an interstitial pneumonia which is histologically similar to viral pneumonia and bronchiolitis in children . Furthermore, the virus biochemically and physically resembles parainfluenza virus type 1, a major pediatric pathogen, and is in the same family as respiratory syncytial virus . Recent publications suggest that flow cytometric analysis of bronchoalveolar lavage (BAL) cells may provide a simple method of quantitating the airway component of the immune response after respiratory infection ."" We decided, therefore, to analyse the sequential changes in the cells recovered by BAL in order to investigate the pathogenetic mechanisms involved in the increased susceptibility to Sendai virus infection in malnourished mice . Results We repeated the central experiments three times with almost identical results . Because there were minor variations between experiments in the ages of the mice (3 weeks versus 5 weeks) and doses of virus [1 x 103 plaque forming units (pfu) versus 2 x 10 3 pfu], we describe mainly the results of a single experiment performed on 3-week-old mice with a 1 x 103 pfu inoculum . As in previous experiments,' mice on the 2% protein diet failed to gain weight during the course of the 2-week preparatory period . At the time of inoculation, proteindeprived mice were entirely normal in appearance and behavior but weighed, on average, 55% of the normally fed mice . Most mortality occurred between days 5 and 9, and exaggerated mortality in the 2% group was observed between days 5 and 8 . As in our previously published work, the overall mortality of the 2% diet group was 87% by the Kaplan-Meier method, whereas that of the 20% protein diet group was 32% (Fig . 1) . We also found, as in previous
100 80 0 60 5
N
40 20 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time after viral inoculation (days)
Fig . 1 . Survival rate of BALB/c mice after intranasal inoculation of Sendai virus, 1 x 103 pfu per animal (Kaplan-Meier method) . Three to five animals were killed every other day for analysis of bronchoalveolar lavage fluid cells . In this experiment 51 mice fed a 20% protein diet for two weeks before inoculation weighed 17 .8±1 .2 g (mean±SD) on day 0 ; 49 were inoculated for the experiment . Seventy-eight mice fed a 2% protein diet for the same interval weighed 9 .8±1 .2 g on day 0 ; all 78 were inoculated for the experiment . 0 : 20% protein diet; 0 : 2% protein diet .
Sendai virus in malnourished mice
1 51
Fig . 2 . Pulmonary virus titer in BALB/c mice with intranasal Sendai virus infection . The lungs were homogenized after bronchoalveolar lavage for analysis of cells . Each point represents mean±SD of three to five mice . The circle at day 9 (2% protein diet group) represents the titer from a single mouse (of five sacrificed in that group) in which virus was grown . Titers are plotted as loglo pfu/g of mouse lung . o : 20% protein diet ; 0 : 2% protein diet .
experiments, that pulmonary virus titers were significantly higher and lasted longer in the malnourished mice (P
