S91

Mechanisms of Action of Intravesical Bacille Calmette-Guerin: Local Immune

Mechanisms
Stephen Prescott, Andrew M. Jackson, Department of Surgery, University of Edinburgh,
Simon J. Hawkyard, Anton B. Alexandroff, Edinburgh, Scotland
and Keith James

The local immune response to mycobacteria is complex, but mycobacterial antigen presen-
tation by phagocytes to T helper cells is the pivotal interaction. Bacille Calmette-Guerin (BCG)
vaccination is associated with the development of antituberculosis immunity but not neces-
sarily with antitumor immunity. Animal studies have shown that an intact host immune system
is required for the antitumor activity of BCG. Immunosuppressed and, particularly, T

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celldepleted individuals fail to respond to BCG immunotherapy. Clinical and laboratory
evidence suggest that the antitumor activity is concentrated at the site of BCG administration,
which reinforces the view that local immune mechanisms are responsible for this phenomenon.

Studies of the immunological mechanism of BCG therapy
show that an intact immune system, particularly the cellular
system, is required for antitumor activity. The administration
of T cells to athymic animals has been shown to restore their
ability to respond to BCG therapy [1]. Clinical and laboratory
evidence show that BCG interaction with the immune system
produces systemic immunity to BCG. The antitumor activity
of BCG appears to be a local phenomenon confined to the site
of administration [2, 3].
The local immune response to mycobacteria is complex, but
mycobacterial antigen presentation by phagocytes to T helper
cells is the pivotal interaction. The presentation of antigen to
T cells requires a series of cell surface interactions, such as
processed antigen and T cell receptor, class II major histocom-
patibility complex (MHC) antigen and CD4, lymphocyte func-
tion antigen 1 (LFA-1) and intercellular adhesion molecule 1
(ICAM-1), and CD28 and CD80. During and after intravesical
BCG therapy, the bladder cancer cell takes on some of the
features of a BCG-infected phagocyte. This may be an impor-
tant factor for tumor cell killing.
Immunohistochemical assessment of serial cold cup bladder
biopsies from patients with carcinoma in situ who are under-
going intravesical BCG therapy shows that there is, in addition
to the obvious polymorphonuclear cell presence, a significant
increase in the density of the mononuclear cell infiltrate in the
lamina propria for 36 months after therapy has been com-
pleted [4, 5]. This is dominated by T cells, particularly of the
CD4 phenotype, and macrophages. The inflammatory response
is associated with change in the phenotype of the bladder tumor
cells (table 1). Before BCG therapy, bladder tumor cells express
Reprints or correspondence: Dr. Stephen Prescott, St. Jamess University
Hospital, Beckett St., Leeds, England (steveprescott@bizonline.co.uk).
Clinical Infectious Diseases 2000 31(Suppl 3):S913
q 2000 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3103S3-0009$03.00
the class I MHC antigen (HLA-ABC), but the class II MHC
molecule (HLA-DR) is expressed weakly or not at all. ICAM-
1 and ICAM-2 are not expressed. After BCG therapy, the blad-
der cancer cells express HLA-ABC, HLA-DR, and ICAM-1
[46].
This change in phenotype has been shown to have a signif-
icant impact on the function of tumor cells. IFN-g produced
by the T cell response to BCG seems to be a key cytokine for
producing the tumor cell phenotypic changes seen in vivo. IFN-
g released in the local immune response to BCG can render
bladder tumor cells capable of acting as both lymphokine-ac-
tivated killer (LAK) cell sensitive targets and antigen presenting
cells for BCG.
The proximity of the bladder wall to the urine has allowed
for an indirect assessment of immune activity in this area by
measurement of cytokine levels in the urine. Several groups of
investigators, including researchers at the University of Edin-
burgh, have examined BCG-induced cytokines both qualita-
tively and quantitatively [710]. They have observed the fol-
lowing: (1) significant increases to biological important levels
of the proinflammatory cytokines IL-1, IL-2, IL-6, IL-8, and
IL-12, IFN-g, and TNF-a; (2) no detectable levels of the Th2
cytokine IL-4, whose role is to terminate the cellular immune
response; and (3) the secretion of a soluble form of ICAM-1
after all instillations in which cytokines have been detected (the
significant secretion has occurred during the first 12 h after
BCG instillation). This in vivo information about the local
immune response to intravesical BCG has been applied to hu-
man bladder cancer cell lines to provide an in vitro model of
BCG treatment of bladder cancer (table 2). The panel of cell
lines were RT4 and UM-UC-3 (low grade); RT112 and 5637
(intermediate grade); and MGH-UI, EJ18, J82 and SD (high
grade). The surface antigen expression of the cell lines differed
from fresh tumor cells because ICAMs were spontaneously ex-
pressed, with ICAM-2 alone on both low grade tumor cell lines
and ICAM-1 alone on all the remaining cell lines [11].
S92 Prescott et al. CID 2000;31 (Suppl 3)

Table 1. Surface phenotype of bladder cancer cells in vivo. ularly with the low and intermediate grade cell lines, but IL-
Antigen Constitutive After BCG la has no effect. At higher doses, IFN-g is actually cytotoxic
Class I MHC (HLA-ABC) 1 1 to RT4 and RT112 [18, 19].
Class II MHC (HLC-DR) 2/1 1 There is a tendency to assume that the bladder tumor cells
ICAM-1 2 1
are passive bystanders in the immune response to BCG, but
ICAM-2 2 2
there is mounting evidence that these cells are very active in
NOTE. ICAM, intercellular adhesion molecule; MHC, major histocompat-
ability complex; 1, expressed; 2, not expressed; 2/1, expressed in a minority
several ways. First, it has been shown [20] that BCG organisms
of cases. in vitro and in vivo bind to and, in some cases, are phagocytosed
by bladder cancer cells. This is an energy-dependent process
that can be inhibited by cell membrane poisons. There is also
Researchers were able to induce or enhance the expression
the suggestion that tumor cells are capable of degrading the
of both HLA-DR and ICAM-1 but not ICAM-2 on the cell
BCG organisms and behave as if they were professional phago-
lines by adding IFN-g alone or, to a lesser extent, TNF-a or
cytes, such as macrophages. Lattime et al. [16] also show that

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IL-la to the culture medium in a dose dependent fashion [12,
tumor cells are capable of antigen presentation, another func-
13]. These effects were also seen if urine from BCG-treated
tion normally ascribed to macrophages, and are also secretors
patients were added to the cell culture medium and could be
of cytokines. Unpublished results from work at the University
largely abolished by the preincubation of the urine with
of Edinburgh show that the majority of TCC cell lines secrete
antiIFN-g antibody [14].
IL-1a and/or IL-6, IL-8, and IL-10 under normal culture con-
The functional importance of the tumor cell phenotypic
ditions (table 3). These researchers believe that IL-la behaves
changes observed in vivo is illustrated in 2 studies [15, 16]. These
as an autocrine factor responsible for constitutive ICAM-1 ex-
studies show that LAK cells are important mediators of the
pression [21].
antitumor response that kills tumor cells in an MHC-unre-
These functions strongly suggest that tumor cells are very
stricted fashion and are probably effector cells in the BCG-
active in the immune responses occurring in the bladder wall
induced antitumor response. LAK cells generated by stimula-
after BCG therapy. The possibility therefore exists that the
tion of peripheral blood lymphocytes with IL-2 and TNF-a in
BCGtumor cell interaction is as important as the BCG-im-
vitro show significant activity against bladder tumor cell targets
munocompetent cell interaction.
[15]. Binding to the target is a requirement, which is achieved
The question that must be asked, then, is what happens if the
by interaction between the LFA-1 molecule on the LAK cell
tumor cell lines are incubated with BCG organisms alone? In
and ICAMs on the tumor cells.
their examination of this question, researchers have observed a
These researchers have found conjugates between T cells that
number of effects on tumor cell proliferation in all lines tested
express LFA-1 and transitional cell carcinoma (TCC) cells that
[22]. First, live BCG and, to a lesser but still significant extent,
express ICAM-1 in the urine of BCG treatment patients. These
heat-inactivated BCG suppress tumor cell growth in a dose-de-
findings support the clinical importance that these phenotype
pendent manner. It has been shown that BCG is not directly
changes. In the TCC cell line model, researchers also have found
cytotoxic to tumor cells in vitro. In addition, the supernatant
that the degree of spontaneous LAK killing of a tumor cell line
from heat-killed BCG does not have an effect on tumor cells.
directly correlates with the level of tumor cell surface expression
Second, researchers at the University of Edinburgh have observed
of ICAM-1 [17]. Moreover, in a given cell line, there is a direct
that the interaction between tumor cells and BCG results in the
relationship between the level of upregulation of expression of
enhanced ICAM-1 expression by the high grade, but not low
ICAM-1 by IFN-g stimulation and the level of LAK killing
grade, TCC cell lines (unpublished data). This effect cannot be
[17]. This enhanced killing can be partially abrogated by the
explained by the autocrine stimulatory effects of TCC cell pro-
use of antiICAM-1 antibodies and, more completely, by the
duced by IL-la and may be due to direct effects of the interaction
use of antiLFA-1 antibodies [17]. In a murine model of bladder
on the gene pool [21]. Third, BCG stimulation results in changes
cancer developed by Lattime et al. [16], the MB49 cell line was
in the cytokine output by TCC cell lines (table 3).
shown to be capable of presenting BCG antigen to BCG-spe-
cific CD4 T cells only when it had been induced to express
HLA-DR by culture with IFN-g. The researchers did not look Table 2. Surface antigen expression by transitional cell carcinoma
for ICAM expression. These studies show that the IFN-g re- cell lines in vitro.
leased in the local immune response to BCG can render bladder Cell line RT4 UMUC3 RT112 5637 SD EJ18 J82 MGHU1
tumor cells capable of acting as both LAK cell sensitive targets Tumor grade G1 G1 G2 G2 G3 G3 G3 G3
and antigen presenting cells for BCG. MHC class I 1 1 1 1 1 1 1 1
MHC class II 1 2 2 2 2 2 2 2
The cytostatic properties of bladder tumor cells may be af- ICAM-1 2 2 ND 2 2 2 2 2
fected by other properties of cytokines present in the post-BCG ICAM-2 1 1 2 2 2 2 2 2
inflammatory response. Both INF-g and TNF-a suppress tu- NOTE. ICAM, intercellular adhesion molecule; MHC, major histocompat-
mor cell growth in vitro in a dose-dependent manner, partic- ability complex; ND, no data; 1, expressed; 2, not expressed.
CID 2000;31 (Suppl 3) Mechanisms of Action of Intravesical BCG S93

Table 3. Constitutive secretion of cytokines by bladder cancer cell 8. Bohle A, Nowc C, Ulmer AJ, et al. Elevations of cytokines interleukin-1,
lines. interleukin-2, and tumor necrosis factor in the urine of patients after
intravesical bacillus Calmette-Guerin immunotherapy. J Urol 1990; 144:
Cytokine RT4 UMU C3 RT112 MGH U1 EJ18 J82 SD 5637
5964.
IL-1a 1 1 1 ND 1 1 1 1 9. De Boer EC, De Jong WH, Steerenberg PA, et al. Induction of interleukin-
IL-6 ND ND ND ND 1 1 1 ND
1 (IL-1), IL-2, IL-6 and tumour necrosis factor during intravesical im-
IL-8 ND ND ND ND 1 1 1 1
munotherapy with bacillus Calmette-Guerin superficial bladder cancer.
IL-l0 ND ND 1 ND 1 ND 1 1
Cancer Immunol Immunother 1992; 34:30612.
NOTE. ND, no data; 1, secretion. 10. Jackson AM, Alexandrov AB, Gribben SC, Esuvaranathan K, James K.
Expression and shedding of ICAM-1 in bladder cancer and its immu-
Conclusion notherapy. Int J Cancer 1993; 55:9215.
11. Jackson AM, Alexandrov AB, Prescott S, James K, Chisholm GD. Role of
This discussion suggests that the effects of BCG on the im- adhesion molecules in lymphokine-activated killer cell killing of bladder
mune system, although many and varied, may act on tumor cancer cells: further evidence for a third ligand for leucocyte func-
cells from 2 main directions, from either (1) the actions on the tionassociated antigen 1. Immunology 1992; 76:28691.

Downloaded from http://cid.oxfordjournals.org/ at Promedica Health System on June 25, 2015

immune system that, from other studies, seem to require live
BCG organisms or (2) the direct actions on tumor cells that
may be activated with dead BCG. Therefore, we propose that
both of these mechanisms are important for the antitumor re-
sponse and that a deficiency in either may result in treatment
failure. Enhanced tumor cell interactions with the immuno-
competent cells in the bladder wall via upregulation of expres-
sion of surface MHC and ICAM antigen is an important factor
for the success of therapy.
REFERENCES
1. Ratliff TL, Gillen D, Catalona WJ. Requirement of a thymus-dependent
immune response for BCG-mediated antitumor activity. J Urol 1987; 137:
1558.
2. Bast RC Jr, Zbar B, Borsos T, Rapp HJ. BCG and cancer. I. N Engl J Med
1974; 290:141320.
3. Bast RC Jr, Zbar B, Borsos T, Rapp HJ. BCG and cancer. II. N Engl J Med
1974; 290:145869.
4. Prescott S, James K, Busuttil A, Hargreave T, Chisholm GC, Smyth J. HLA-
DR expression by high grade superficial bladder cancer treated with BCG.
Br J Urol 1989; 63:2649.
5. Prescott S, James K, Hargreave TB, Chisholm GD, Smyth JF. Intravesical
Evans strain BCG therapy: quantitative immunohistochemical analysis of
the immune response within the bladder wall. J Urol 1992; 147:163642.
6. Jackson AM, Alexandroff AB, McIntyre M, Esuvaranathan K, James K,
Chisholm GD. Induction of ICAM-1 expression on bladder tumours by
BCG immunotherapy. J Clin Pathol 1994; 47:30912.
7. Prescott S, James K, Hargreave TB, Chisholm GD, Smyth JF. Radio-
immunoassay detection of interferon-g in urine after intravesical Evans
BCG therapy. J Urol 1990; 144:124851.
12. Jackson AM, Alexandrov AB, Prescott S, James K, Chisholm GD. Expres-
sion of adhesion molecules by bladder cancer cells: modulation by inter-
feron-g and tumor necrosis factora. J Urol 1992; 148:15836.
13. Hawkyard S, James K, Prescott S, et al. The effects of recombinant interferon-
g on a panel of human bladder cancer cell lines. J Urol 1991; 145:107881.
14. Jackson AM, Prescott S, Hawkyard K, James K, Chisholm G. The immu-
nomodulatory effects of urine from patients with superficial bladder cancer
receiving intravesical Evans BCG therapy. Cancer Immunol Immunother
1993; 36:2530.
15. Jackson AM, Hawkyard S, Prescott S, Ritchie AWS, James K, Chisholm
GD. An investigation of factors influencing the in vitro induction of LAK
activity against a variety of human bladder cancer cell lines. J Urol 1992;
147:20711.
16. Lattime EC, Gomella LG, McCue PA. Murine bladder carcinoma cells pre-
sent antigen to BCG-specific CD41 T cells. Cancer Res 1992; 52:428690.
17. Jackson AM, Alexandroff A, James K. The requirement of adhesion mol-
ecules for cell mediated tumour cell destruction. In: Epenetos AA, Pig-
natelli M, eds. Cell adhesion molecules in cancer and inflammation. New-
ark, NJ: Harwood Academic Publishers, 1995:13346.
18. Hawkyard S, Jackson AM, James K, Prescott S, Smyth JF, Chisholm GD.
The growth inhibitory effects of interferon-g on bladder cancer cells.
J Urol 1992; 147:1399403.
19. Hawkyard SJ, Jackson AM, Prescott S, James K, Chisholm GD. The effect
of recombinant cytokines on bladder cancer cells in vitro. J Urol 1993;
150:5148.
20. Becich MJ, Carroll S, Ratliff TL. Internalization of bacille Calmette-Guerin
by bladder tumor cells. J Urol 1991; 145:131624.
21. Alexandroff AB, Jackson AM, Esuvaranathan K, Prescott S, James K. Au-
tocrine regulation of ICAM-1 expression on bladder cancer cell lines:
evidence for the role of IL-la. Immunol Lett 1994; 40:11724.
22. Jackson AM, Alexandroff AB, Fleming D, Prescott S, Chisholm GD, James
K. Bacillus Calmette-Guerin (BCG) organisms directly alter the growth
of bladder tumor cells. Int J Oncol 1994; 5:697703.