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Bronchoalveolar Lavage Fluid and Handling of Mycobacteria
Bronchoalveolar Lav. Fluid arid Handling of Mycobacterla* Rodney P. Swartz, Ph.D.; and Henry Yeager; Jr.. M.D.• F.C.C.P.
I
n the alveolar spaces of the lung, lDvading mycobacteria encounter an environment of aeellular mlrterial derived from lymphocytes, alveolar macrophages, and ~ther lung cells. Such acellular material may influence the I10st phagocyte disposition of mycobacteria. In in vitro cultures, we utilized human autologous alveolar macrophages (AAM). peripheral blood monocytes (PBM). and bronchoalveolar lavage fluid (BALF) obtained from healthy volunteers after informed consent was obtained. The AAM and PBM were adhered to 12-mm coverslips for uptake assays or into multiple wells for mycobactericidal assays. The BALF was membrane-Bher-concentrated at 4°C to a protein content of 1 mg/ml before being prepared as (a) untreated whole BALF, (b) heated to ~C for 1 h. or (c) decoinplemented with cobra venom factor. Th determine whether BALl<' directly affected phagoeytes, or whether ally alteration was due to opsonic activity, BALF was either added directly to the medium. or the Mycobacteria iJofum complex (MAC) organisms were suspended in the BALF (2.5 x 10'"/ml) at 3T'C. 1 h. then washed in Hanks balanced salt solution. suspended in medium. lind added to the AAM or PBM cultures. No direct activation of the phagocytes occurred. Only when MAC organisms were preexposed to BALF was there significilntly enhanced uptake of MAC by both AAM and PBM . Complement was a major component of the BALF effect on uptake. In contrast. HALF reduced the killfug of mycobacteria by both AAM and PBM . Decomplementing or heating the BALF reduced any myobactericidal activity even further. Next, we utilized an in vivo murine model to assess complement's significance in mycobacterial infection of the lung. hi 2 separate experiments, C57BlI6 mice were decemplemented with cobra venom factor and then infected intravenously witli M aoium complex organisms. No viable mycobacteria were recovered from the lungs of control mice. but decomplemented mice had significantly more mycobacteria present in their lungs (Exp A. 1.067 ± 500 cfu; Exp B. 1,500 ± 500 cfu). In conclusion. extracellular alveolar materials (especially' complement) may play a major role in the handling of mycobacteria by macrophages in vitro and probably. in vivo. -From Georgetown University Medical Center, Washington. DC.
HostDefenses Against '. Pneumocvstls carln/lln Mice selectlvery Depleted of CD4+ lymphocytes*
rzrz:
}tmIu M. Bide, M.D.• F.C.C.P.; Martha L. "Umock. M.D.; H.1ienfrr KtJltMder; M.D., F.C.C.P.; andJudd E. SheUUo, M.D.
carinii pneumonia is an important infectious complication of AIDS. but pulmonary host defenses against this pathogen are poorly understood . I •• Depletion of CD4 + lymphocytes, which is the major immunologic abnormality in patients with AIDS. is clearly correlated with an increased risk of development of P carinii pneumonia," However. the specific role of the CD4+ lymphocyte in host defense against P carinU requires further investigation. The CD4 + lymphocyte is unlikely to function alone as ail effector cell against P carinii. Instead. its role in host defense may be to produce activation signals that influence other inflammatory cells. Failure of CD4 + lymphocytes to produce these activation signals may result in the establishment of P carinii infection. For example. the inabilitY of CD4 + lymphocytes to produce interferon-gamma (IFN-'Y) could result in de6cient activation of alveolar macrophages. Indeed. lymphocytes obtained from patients with AIDS are de6cient in their ability to elaborate IFN-'Y.· but alveolar macrophages from these patients can be activated normally in vitro by exogenous IFN-'Y.a In addition to IFN-oy. other cytokines produced by CD4 + lymphocytes may also be important in modulation of host defense against P carinii . Because P carinii can only be maintained in short-term culture. animal models have been used to examine host defense against the organism. A model utilizing corticosteroid immunosuppression of rodents has been utilized extensively,· but the multiple immunologic defects induced by corticosteroids complicate the interpretation of data concerning host defense. Recent reports of spontaneous P carin;; infection in mice with severe combined immunode6ciency promise to provide Important data concerning host defense,".• In this environmentally-acquired model of infection. however. the time course after acquisition of P carinii cannot be studied. and the inoculum size cannot be manipulated experimentally. REQUIREMENT FOR
CD4+
LYMPHOCYTES IN
DEFENSE AGAINST
HoST
P CAlllNll
To test the hypothesis directly that CD4 + lymphocytes are crucial for host defense against P carinii. we developed and characterized a model of P carinii infection in mice specifically depleted of crx + lymphocytes," The goal was to establish an infection that is reproducible. persistent. and associated with a discrete immune defect mirroring the -From the Pulmonary and Critical Oare Medicine Section, Veterans Affairs Medical Center, and the Departments of Medicine (Drs. Beck and ICaltreider) and Pathology (Dr. Warnock), University of CaliforiJia, San Francisco; and the Section of Pulmonary and Critical Care Medicine (Dr. Shellito), Louisiana State University Medical Center, New Orleans. These studies were supported by funds from the Department of Vetenlns Affairs, by the Am«;rican Lung Association of California, atld by NIH grants HL-29246 and HL-34298. Reprint requem: Dr. Beck, l\Jmonary Medicine(111D). VA Medical Center; 4150 Clement. San FrancUco 94121-1598
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I
n the alveolar spaces of the lung, lDvading mycobacteria encounter an environment of aeellular mlrterial derived from lymphocytes, alveolar macrophages, and ~ther lung cells. Such acellular material may influence the I10st phagocyte disposition of mycobacteria. In in vitro cultures, we utilized human autologous alveolar macrophages (AAM). peripheral blood monocytes (PBM). and bronchoalveolar lavage fluid (BALF) obtained from healthy volunteers after informed consent was obtained. The AAM and PBM were adhered to 12-mm coverslips for uptake assays or into multiple wells for mycobactericidal assays. The BALF was membrane-Bher-concentrated at 4°C to a protein content of 1 mg/ml before being prepared as (a) untreated whole BALF, (b) heated to ~C for 1 h. or (c) decoinplemented with cobra venom factor. Th determine whether BALl<' directly affected phagoeytes, or whether ally alteration was due to opsonic activity, BALF was either added directly to the medium. or the Mycobacteria iJofum complex (MAC) organisms were suspended in the BALF (2.5 x 10'"/ml) at 3T'C. 1 h. then washed in Hanks balanced salt solution. suspended in medium. lind added to the AAM or PBM cultures. No direct activation of the phagocytes occurred. Only when MAC organisms were preexposed to BALF was there significilntly enhanced uptake of MAC by both AAM and PBM . Complement was a major component of the BALF effect on uptake. In contrast. HALF reduced the killfug of mycobacteria by both AAM and PBM . Decomplementing or heating the BALF reduced any myobactericidal activity even further. Next, we utilized an in vivo murine model to assess complement's significance in mycobacterial infection of the lung. hi 2 separate experiments, C57BlI6 mice were decemplemented with cobra venom factor and then infected intravenously witli M aoium complex organisms. No viable mycobacteria were recovered from the lungs of control mice. but decomplemented mice had significantly more mycobacteria present in their lungs (Exp A. 1.067 ± 500 cfu; Exp B. 1,500 ± 500 cfu). In conclusion. extracellular alveolar materials (especially' complement) may play a major role in the handling of mycobacteria by macrophages in vitro and probably. in vivo. -From Georgetown University Medical Center, Washington. DC.
HostDefenses Against '. Pneumocvstls carln/lln Mice selectlvery Depleted of CD4+ lymphocytes*
rzrz:
}tmIu M. Bide, M.D.• F.C.C.P.; Martha L. "Umock. M.D.; H.1ienfrr KtJltMder; M.D., F.C.C.P.; andJudd E. SheUUo, M.D.
carinii pneumonia is an important infectious complication of AIDS. but pulmonary host defenses against this pathogen are poorly understood . I •• Depletion of CD4 + lymphocytes, which is the major immunologic abnormality in patients with AIDS. is clearly correlated with an increased risk of development of P carinii pneumonia," However. the specific role of the CD4+ lymphocyte in host defense against P carinU requires further investigation. The CD4 + lymphocyte is unlikely to function alone as ail effector cell against P carinii. Instead. its role in host defense may be to produce activation signals that influence other inflammatory cells. Failure of CD4 + lymphocytes to produce these activation signals may result in the establishment of P carinii infection. For example. the inabilitY of CD4 + lymphocytes to produce interferon-gamma (IFN-'Y) could result in de6cient activation of alveolar macrophages. Indeed. lymphocytes obtained from patients with AIDS are de6cient in their ability to elaborate IFN-'Y.· but alveolar macrophages from these patients can be activated normally in vitro by exogenous IFN-'Y.a In addition to IFN-oy. other cytokines produced by CD4 + lymphocytes may also be important in modulation of host defense against P carinii . Because P carinii can only be maintained in short-term culture. animal models have been used to examine host defense against the organism. A model utilizing corticosteroid immunosuppression of rodents has been utilized extensively,· but the multiple immunologic defects induced by corticosteroids complicate the interpretation of data concerning host defense. Recent reports of spontaneous P carin;; infection in mice with severe combined immunode6ciency promise to provide Important data concerning host defense,".• In this environmentally-acquired model of infection. however. the time course after acquisition of P carinii cannot be studied. and the inoculum size cannot be manipulated experimentally. REQUIREMENT FOR
CD4+
LYMPHOCYTES IN
DEFENSE AGAINST
HoST
P CAlllNll
To test the hypothesis directly that CD4 + lymphocytes are crucial for host defense against P carinii. we developed and characterized a model of P carinii infection in mice specifically depleted of crx + lymphocytes," The goal was to establish an infection that is reproducible. persistent. and associated with a discrete immune defect mirroring the -From the Pulmonary and Critical Oare Medicine Section, Veterans Affairs Medical Center, and the Departments of Medicine (Drs. Beck and ICaltreider) and Pathology (Dr. Warnock), University of CaliforiJia, San Francisco; and the Section of Pulmonary and Critical Care Medicine (Dr. Shellito), Louisiana State University Medical Center, New Orleans. These studies were supported by funds from the Department of Vetenlns Affairs, by the Am«;rican Lung Association of California, atld by NIH grants HL-29246 and HL-34298. Reprint requem: Dr. Beck, l\Jmonary Medicine(111D). VA Medical Center; 4150 Clement. San FrancUco 94121-1598
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