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From the Trudeau Institute, Saranac Lake, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD40-CD154 interaction is a well known critical component in the activation of adaptive immune responses.19,20 Of potentially equal importance, but less well defined, is the in vivo role of CD40-CD154 interaction in innate immunity and its association with the development of immune-mediated inflammation. In vitro activation of endothelial cells and lung fibroblasts by CD40 ligation results in the up-regulated expression of cellular adhesion molecules and increased production of proinflammatory cytokines.21,22 Ligation of CD40 on human monocytes in vitro results in enhanced tumoricidal activity, induction of a state of activation, and production of proinflammatory cytokines.7 These findings have led to speculation that CD40+ cells contribute to the activation and regulation of immune-mediated inflammation.23 The extent to which these in vitro findings have been extrapolated to in vivo models has remained incomplete. In vivo modeling of CD40 involvement in inflammation is also warranted by the potential for application of a CD154 plasmid construct as a transgenic adjuvant in vaccination therapies.24 Consideration must be given to the nonspecific stimulation of unrelated CD40+ cells that encounter the CD154-transfected cells. The consequences of unrestricted CD40-CD154 interactions have been demonstrated in a CD154 transgenic mouse model.25 In vivo models to study the role of CD40-CD154 interactions in the establishment and maintenance of inflammation are needed to further understand how this interaction could impact immune-mediated inflammatory diseases.
We have recently shown that instilling soluble CD154 into the lungs of mice results in an inflammatory response similar to that which occurs during human pulmonary immune responses.26 Our in vivo model of CD40 ligation is useful for the investigation of immune-mediated pulmonary inflammation. In this paper, we report on the involvement of different CD40+ cells in establishing and maintaining a pulmonary inflammatory response induced by a specific anti-CD40 monoclonal antibody (mAb). Our evidence demonstrates that CD40+ BM-derived cells in the lung (macrophages, B cells, and possibly dendritic cells) were critical to the establishment of this response, but were not by themselves capable of sustaining the full extent of the inflammation. CD40+ non-BM-derived cells of the lung (endothelial cells, epithelial cells, and fibroblasts) were not capable of establishing this response in the absence of CD40+ BM-derived cells. In the absence of B cells, macrophage recruitment and pulmonary edema associated with this CD40 ligation-dependent inflammation were diminished; they were diminished further in the absence of B and T cells. The results indicate that CD40+ BM-derived cells were critical to the establishment of CD40-induced pulmonary inflammation and that CD40+ non-BM-derived cells of the lung were also required to maximize the inflammatory response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female C57BL/6 wild-type (+/+) mice, approximately 9 weeks of age, were obtained from the Trudeau Institute animal breeding facility for use in this study. CD40-/- breeder pairs were obtained from Dr. R. Geha27 and bred at the Trudeau Institute animal breeding facility. Male CD40 knockout (-/-) mice used in this study were approximately 10–12 weeks of age. Breeding pairs of µMT mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at the Trudeau Institute breeding facility. Male µMT mice used in this study were approximately 10–12 weeks of age. Ten-week-old male SCID mice on a C57BL/6 background were obtained directly from The Jackson Laboratory for this study.
Production of Anti-CD40 Antibody
The hybridoma cell line which secretes the rat IgG anti-CD40 mAb, 1C10, was obtained by permission from DNAX Corporation (Palo Alto, CA). The cell line was cultured in RPMI 1640 media (Gibco BRL, Grand Island, NY) supplemented with 3–10% fetal calf serum, 200 U/ml penicillin, 200 ug/ml streptomycin, and 20 mmol/L HEPES buffer. The cells were grown in a CellMax artificial capillary cell culture system (Spectrum Labs, Laguna Hills, CA) for three months, during which time tissue culture supernatant was recovered regularly. The antibody was purified using an AvidChrom IgPure column (Unisyn Technologies, Hopkinton, MA). The control mAb used in these procedures was a rat IgG mAb specific for horseradish peroxidase (HRPN) which was grown and purified in the same manner as the anti-CD40 mAb.
Chimeric Mice
Chimeric mice were made by reconstituting the BM compartment of +/+ or CD40-/- mice following irradiation. Recipient mice were irradiated (9.5 Gy) and then immediately reconstituted by i.v. injection of 107 +/+ or CD40-/- donor BM cells. The animals were then housed for 1 month, under sterile conditions when required, to allow re-establishment of their BM compartment. After this time, peripheral blood samples were taken from the reconstituted animals. B cells were stained for the presence or absence of CD40 using FITC-anti-CD40 and phycoerythin-anti-B220 (PharMingen, San Diego, CA) and analyzed by flow cytometry.
Experimental Design
Anti-CD40 mAb was given in 60-ug doses by intratracheal administration to mice under light halothane anesthesia. The mAb was given daily for 3 consecutive days and then every second day for 6 days. The mice were then exsanguinated by perforation of the abdominal aorta following deep halothane anesthetization. The difference between the starting and final body weight of each mouse was recorded as the change in body weight over the duration of the experiment. The lungs of the mice were lavaged as previously described.28 Briefly, the trachea was exposed, cannulated, and washed incrementally with five 1-ml volumes of Hanks' balanced salt solution:EDTA (3 mmol/L). Using this lung lavage technique, at least 85% of the lavage fluid was retrieved. The total number of cells recovered in the lung lavage fluid was recorded and an aliquot of the cells obtained from each animal was stained using Diff-Quik (Baxter, Miami, FL). Measurements of the serum albumin levels found in the lung lavage fluids have been used as indicators of tissue injury and subsequent transudation of proteins from the bloodstream.29 Serum albumin levels found in the lung lavage fluids were determined using an albumin colorimetric assay (Sigma Diagnostics, St. Louis, MO). Color absorbency was read at 630 nm and albumin concentration was reported as ug/ml of lung lavage fluid. Lung tissue sections were cut from lungs fixed in phosphate-buffered 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin for histological assessment.
Flow Cytometry
Cells obtained from lung lavage were stained with the following fluorochrome-conjugated mAbs: phycoerythrin-anti-B220, cychrome-anti-CD4, biotinylated-anti-CD8, phycoerythrin-anti-CD44 (PharMingen, San Diego, CA), streptavidin-allophycocyanin (Caltag Laboratories, Burlingame, CA), FITC-anti-IgG, A, M (Organon Teknika, West Chester, PA), and FITC-anti-CD62L (Trudeau Institute, Saranac Lake, NY). The cells were washed and resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin-0.1% sodium azide. The cells were stained for 30 minutes at 4°C. Confinement of the analysis to the lymphocyte population was accomplished by establishing forward and side scatter settings to exclude other cell populations. Surface marker phenotypes were detected on a FACS caliber cytofluorometer (Becton Dickinson, San Jose, CA) and analyzed using CellQuest software (Becton Dickinson).
Statistics
Data are expressed as the mean ± standard deviation (SD). The results reported here are from one experiment that was representative of two independent experiments. The sample size of each group is as stated for each experiment. Differences between the designated groups were determined using Mann-Whitney rank sum analysis or a pairwise Dunn's ANOVA on the difference of rank means. Differences were considered significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The specificity of the anti-CD40 mAb and its ability to induce a
pulmonary inflammatory response are shown (Table 1)
. Treatment of +/+ mice with anti-CD40
mAb induced a significant cellular infiltration that was not observed
in similarly treated CD40-/- mice. A substantial accumulation of
cells in the alveolar space as well as significant levels of
perivascular and peribronchiolar cuffing were evident in the +/+ mice
treated with anti-CD40 mAb (Figure 1a)
.
Treatment of CD40-/-mice with anti-CD40 mAb (Figure 1b)
and treatment
of +/+ mice (Figure 1c)
and CD40-/- mice with anti-HRPN mAb elicited
none of the above indications of pulmonary histopathology. A
significant transudation of serum albumin into the alveolar space
(Figure 2a)
and the development of a
wasting syndrome characterized by body weight loss (Figure 2b)
were
observed only in the +/+ mice treated with anti-CD40 mAb. In fact, a
slight weight gain was observed over the duration of the experiment in
the CD40-/- mice treated with anti-CD40 mAb and in the +/+ mice
treated with the control anti-HRPN mAb.
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Anti-CD40 mAb treatment of the +/+ mice that received +/+
BM-derived cells elicited an intense pulmonary cellular infiltrate
(Table 2)
. Similar treatment of the +/+
or CD40-/- mice given CD40-/- BM-derived cells or of the CD40-/-
mice given +/+ BM-derived cells resulted in reduced pulmonary cellular
infiltration levels. However, the total numbers of lymphocytes and
polymorphonuclear cells (PMNs) recovered from the CD40-/- mice
that received +/+ BM-derived cells were significantly greater than
those recovered from +/+ mice or CD40-/- mice that received CD40-/-
BM-derived cells. Thus, the cellular infiltration was greatest in the
+/+ mice given +/+ BM-derived cells, slightly elevated in the CD40-/-
mice given +/+ BM-derived cells, and nearly undetectable in either the
+/+ mice or CD40-/- mice given CD40-/- BM-derived cells.
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Little detectable lymphocyte infiltration occurred in the mice given
CD40-/- BM-derived cells regardless of whether the recipients were
+/+ or CD40-/- mice. In mice that received +/+ cells, the level of
CD8+ T cell infiltration was greater than that of the CD4+ T cells. In
both the CD4+ and CD8+ T cell subsets, the activation phenotype
CD44high/CD62Llow
predominated.
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. The only other mice to show
detectable albumin levels in their lung lavage fluids were the
CD40-/- mice that received +/+ BM-derived cells. In a similar manner,
only those mice reconstituted with +/+ BM-derived cells showed a
significant loss in body weight (Figure 3b)
.
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Macrophage infiltration into the lungs of anti-CD40 mAb-treated
µMT and SCID mice was substantially reduced relative to similarly
treated +/+ mice (Table 4)
. As expected,
the lymphocyte infiltration level into the lungs of the anti-CD40
mAb-treated SCID mice was significantly reduced compared to the +/+
mice. Although the PMN influx in the anti-CD40 mAb-treated µMT
mice was significantly greater than that for the +/+ mice in this
experiment, the PMN influx in the repeat experiment was equivalent
among the three strains of mice. The levels of serum albumin in the
lung lavage fluids of the treated µMT and SCID mice were both
significantly reduced relative to the similarly treated +/+ mice, with
that for the SCID mice being significantly lower than that for the
µMT mice (Figure 4a)
. The +/+, µMT,
and SCID mice all lost equivalent amounts of body weight (Figure 4b)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The chimeric mouse model differentiates between the levels of
necessity for either the CD40+ BM- or non-BM-derived cells of
the lung in the induction and maintenance of a pulmonary inflammatory
response. The intensity of the cellular influx, body weight loss, and
albumin levels in the lung lavage fluids of +/+ mice that received +/+
BM-derived cells was significantly greater than that observed for +/+
mice that received CD40-/- BM-derived cells as well as for CD40-/-
mice that received either +/+ or CD40-/- BM-derived cells. However,
the lesser intensity of the pathological sequelae observed in the
CD40-/- mice that received +/+ BM-derived cells was still
significantly greater than for the +/+ or CD40-/- mice that received
CD40-/- BM-derived cells. The evidence from these BM chimeras
suggested that CD40+ BM-derived cells such as macrophages, B cells,
and, possibly, dendritic cells were responsible for the establishment
of this inflammatory response. The lesser, albeit significant, response
of the CD40-/- mice that received +/+ BM-derived cells suggested that
CD40+ non-BM-derived cells maximized the response despite being unable
to establish it by themselves. The equivalent lack of intensity of the
inflammatory responses in the +/+ and CD40-/- mice that received
CD40-/- BM-derived cells is consistent with this hypothesis. The +/+
mice that received +/+ BM-derived cells and the CD40-/- mice that
received CD40-/- BM-derived cells responded to the anti-CD40 mAb
treatment in a manner similar to that seen with the +/+ and CD40-/-
mice, respectively (Table 1
versus Table 2
, Figure 2a
versus Figure 3a
). This suggested that the irradiation and
reconstitution procedure did not affect the response to the anti-CD40
mAb treatment.
The actual contribution of the CD40+ non-BM-derived cells of the lung to the inflammatory response may be as a source of required inflammatory mediators such as soluble cytokines or chemokines and adhesion molecules. Ligation of CD40 on fibroblasts and endothelial cells in vitro results in the production of these inflammatory mediators.18,21,22,30 The elicitation of inflammatory mediators from these CD40+ non-BM-derived cells could be reliant on a series of sequential signals. Soluble cytokines and chemokines elicited from CD40-activated BM-derived cells could induce an increase in the level of expression of constitutive CD40 on non-BM-derived cells in the lung.15,16 The enhanced CD40 signaling, in conjunction with that by inflammatory cytokines, may in turn elicit an activated state in the CD40+ non-BM-derived cells. The absence of either of these signaling mechanisms could result in a diminished response or its ablation, depending on which signal is lacking.
In the CD40-/- mice that received +/+ BM-derived cells, direct activation of B cells and indirect T cell activation via CD40-induced macrophage products may account for the recruitment of these cells into the alveolar space. However, the inability to induce a sufficient level of CD40-dependent expression of proinflammatory mediators from the non-BM-derived cells of the lung may have limited their recruitment. The absence of a lymphocytic infiltrate in the mice that received CD40-/- BM-derived cells would suggest the inability either to activate these BM-derived cells or to produce inflammatory mediators from the CD40+ non-BM-derived cells in the lung. In the chimeric mice that received +/+ BM-derived cells, the characteristics of the T cell infiltrates were similar. The number of infiltrating CD8+ T cells was substantially greater than the number of CD4+ T cells. In both the CD4+ and CD8+ subsets, the majority of infiltrating cells exhibited the activation phenotype CD44high/CD62Llow. The significance of this finding is a subject of current investigation.
The observation that significant numbers of B cells and T cells were recovered from the lung lavage fluids of mice that received +/+ BM-derived cells led to the possibility that B cells and T cells may have been actively recruited into and may have contributed to the CD40-induced response, rather than having been bystanders. In µMT and SCID mice, the level of macrophage infiltration following treatment with anti-CD40 mAb was reduced relative to that seen in similarly treated +/+ mice. This suggested that B cells and/or T cells may have been associated with the recruitment of additional macrophages in this response. That the loss of B cells, or of both B cells and T cells, did not result in a decrease in the PMN influx suggested either that PMN recruitment was independent of lymphocyte recruitment or that it preceded lymphocyte recruitment in this response. The production of neutrophil chemoattractants by CD40 activation of resident alveolar macrophages could have been responsible for PMN recruitment. In vitro priming of macrophages by CD40 ligation results in the production of neutrophil chemoattractants and the up-regulation of macrophage CD40 surface expression.16 Increased CD40 expression is also observed on alveolar macrophages recovered from sarcoidosis patients.31 It has been suggested that chemokine products from in vivo primed alveolar macrophages may be directly involved in initial neutrophil recruitment in inflammatory diseases of the lung.32-34 In view of the evidence in the literature, our results suggest that resident and newly recruited alveolar macrophages activated by CD40 ligation were involved in recruiting PMNs into the lung during this immune-mediated pulmonary inflammation.
Evidence from the CD40 chimeras implied that the presence of +/+ BM-derived cells were critical in the induction of pulmonary edema. The significantly reduced serum albumin levels found in the lung lavage fluids of µMT and SCID mice suggested that both B cells and T cells were involved in the edema associated with this response. That CD40-induced edema and PMN infiltration were still induced in SCID mice suggested that a cause-and-effect relationship between these cells and pulmonary damage could be associated with the CD40-mediated activation of alveolar macrophages. The priming of alveolar macrophages is associated with their production and release of neutrophil chemotactic proteins, and the pulmonary transudation of albumin in response to lung trauma.32 Our results comport with the evidence that suggests an active role for primed alveolar macrophages, in conjunction with neutrophil and lymphocyte sequestration, in the establishment and maintenance of CD40-induced pulmonary edema.
As with the development of pulmonary edema, the CD40-induced wasting syndrome was associated with the receipt of +/+ BM-derived cells by either +/+ or CD40-/- mice. However, the lack of any differences in the change in body weight between the +/+, µMT, and SCID mice suggests that T cells and B cells do not influence the CD40-induced wasting syndrome. The CD40-mediated activation of macrophages and possibly the subsequent neutrophil involvement were likely responsible for the associated weight loss in this inflammatory response.
In summary, our CD40 chimeric mouse model of immune-mediated pulmonary inflammation suggests that an induction sequence initially involving CD40+ BM-derived cells and followed by induction of CD40+ non-BM-derived cells is required to establish and maintain the CD40-dependent inflammatory response to its full extent in the lung. The pathological sequelae associated with this response can all be linked to CD40-dependent activation of BM-derived cells. Not all of these sequelae were associated with B cell and T cell activation. However, activation of alveolar macrophages by CD40 ligation is most likely responsible for the events preceding the induction of these sequelae. The level of neutrophil recruitment in the chimeric and knockout models and their association with the intensity of each of the sequelae imply that these cells are required in the inflammatory response. The use of in vivo CD40-induced pulmonary inflammation models establishes a link between the cause and effect seen in in vitro cell activation studies and the pathological observations in vivo with pulmonary inflammation syndromes. Further studies on the involvement of specific cell subsets and their inducible products are underway.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant HL55002L.
Accepted for publication December 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+/+ mice group,
P < 0.05, statistically significant difference
relative to +/+ cells 