I
`
`Clin. exp lmmuno/. (19SS) 62,3i0..:.3!4.
`
`In vivo and in vitro effects of doxycycline on leucocyte
`membrane receptors
`
`A. NiESS, J. GLETTE, A. I. HALSTENSEN, S. SANDBERG & C. 0. SOLBERG
`Medical Department Band Laboratory of Clinical Biochemistry, Haukeland Hospital, University
`of Bergen, Norway
`
`(Accepted for publication 14 May 1985)
`
`SUMMARY
`
`Tetracyclines, particularly doxycycline, have adverse effects on granulocyte function in
`vitro. We have examined the effects of doxycycline on membrane receptors for IgG (Fcy·
`R) and C3b (C3b-R) on granulocytes and lymphocytes, as well as on the sheep erythrocyte
`receptor (E-R) on T lymphocytes. Acne patients given doxycycline orally had a lower
`percentage of Fcy-R positive granulocy.tes (57%) than before treatment (80%) or
`compared to healthy controls (81%). Following in vitro doxycycline incubation, normal
`granulocytes showed decreased levels of Fey-R positive cells. This effect was counteracted
`by the addition of magnesium during incubation. The deleterious effect of doxycycline on
`granulocyte functions may be due to decreased levels of Fcy-R bearing granulocytes.
`Doxycycline in vivo or in vitro had no significant effect on the proportion of C3b-R bearing
`granulocytes or lymphocytes or the T lymphocyte percentage. After in vitro irradiation
`with light at 340-380 nm, however, both granulocytes and lymphocytes preincubated with
`doxycycline showed up to 50% decrease in Fcy-R bearing cells, while control cells without
`doxycycline were unaffected.
`
`Keywords doxycycline Fe receptor C3b receptor
`
`leucocytes
`
`INTRODUCTION
`
`The effects of tetracyclines, and in particular doxycycline, on polymorphonuclear leucocyte (PMN)
`functions have been extensively studied. In vitro studies have shown that doxycycline has
`deleterious effects on PMN migration (Belsheim, Gnarpe & Lafberg, 1979; Glette et al., 1982),
`phagocytosis (Forsgren, Schmeling & Quie, 1974; Gnarpe & Leslie, 1974), chemiluminescence and
`glucose oxidation (Giette eta/., 1982). The results of some, but not all, in vivo studies also indicate a
`deleterious effect (Glette eta!., 1982). In addition, Forsgren & Gnarpe (1973) found doxycycline to
`inactivate the complement system and interfere with the bactericidal effect of serum. Little is known
`about the mechanisms underlying these various effects of doxycycline. In the present study
`doxycycline in vivo and in vitro is shown to depress rosette formation between PMNs and antibody-
`coated erythrocytes, thus implying interference with the receptor for the Fe portion of
`immunoglobulin G.
`
`MATERIALS AND METHODS
`
`Peripheral blood was obtained from eight patients with acne, and from 25 healthy controls. Samples
`from the patients were taken immediately before and I week after institution of200 mg doxycycline
`orally per day. PMNs and lymphocytes were separated as described by Talstad(l981). Briefly, 2 ml
`Correspondence: Dr A. Na:ss, Medical Department B, N-5016 Haukeland, Sykehus, Norway.
`
`310
`
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`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
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`Effects of doxycycline on leucocyte membrane receptors
`311
`of heparinized (18 u/ml) blood was mixed with 2 ml of phosphate-buffered saline (PBS) and
`carefully layered on top of a two-layer mixture of Ficoll (Pharmacia, Stockholm, Sweden) and
`Isopaque (Nyegaard & Co., Oslo, N orway) in a plastic tube. After centrifugation at 600 g for 30 min
`the PMNs and lymphocytes, forming separate layers, were harvested, washed three times in PBS
`and resuspended in PBS to a concentration of 4 x 106/ml.
`Rosette techniques. 'Active' E-rosette-forming ce/ls (active E-RFC) were enumerated by adding
`0·2 ml of 1% sheep erythrocytes (E) to 0·2 ml oflymphocyte suspension, centrifuging at 200 g for 5
`min, resuspending, and counting (Smith et al., 1975). The active E-RFC population is considered to
`represent the immunologically active T lymphocytes (Wybran & Fudenberg, 1973).
`E-rosette-forming cel/s (E-RFC) were determined as described previously (Nress & Nyland,
`1978). A lymphocyte suspension of0·2 ml was mixed with an equal volume of0·5 E, incubated for 5
`min at 37°C, centrifuged for 5 min at 200 g, and stored in ice water overnight before being counted.
`PMN suspensions did not form active E-RFC or E-RFC.
`EA-rosette-forming ce/ls (EA-RFC) were enumerated by an assay with ox E sensitized with I
`agglutinating unit of rabbit lgG antiborlies (A). By this technique the presence on the cell of
`receptors for the Fe portion oflgG (Fcy-R) is demonstrated (Hallberg, Gurner & Coombs, 1973).
`Lymphocytes or PMN were mixed with an equal volume (0·2 ml) of indicator cells, and incubated at
`room temperature for 20 min.
`EAC-rosette-forming cel/s (EAC-RFC) were determined using sheep E sensitized with rabbit
`lgM antiborlies (A). Humanserum absorbed with zymosan was used as the source of complement
`(C). A mixture of 0·2 ml of lymphocyte or PMN suspension and 0·2 ml of EAC was incubated at
`37°C for 20 min. Cells having receptors for the C3b fraction of complement (C3b-R) form rosettes
`by this method (Matre & T0nder, 1976).
`All rosette preparations were counted by the same person, at a magnification of x 600. A
`minimum of 200 cells were counted and those with four or more adhering E were counted as RFC.
`lncubation studies. Sampies of0·2 ml ofPMN or lymphocytes were incubated at 37°C for 30 min
`in PBS, or PBS with doxycycline at the concentration of 6·25, 12·5, 25, 50 and 100 J.tg/ml. After
`incubation, cells were washed three times in PBS, and resuspended to 4 x 106/ml. Rosette sturlies
`were then performed as described above. In an additional experiment, 4 mM of Mg2+ was added to
`incubation tubes containing 50 j.lg doxycycline/ml. Similar experiments using Ca2+ were abandoned
`because of excessive clumping of cells.
`Irradiation. Contaminating erythrocytes in the granulocyte suspension were first lysed with
`ammonium chloride (Weening, Roos & Loos, 1974). The cells were than resuspended in PBS, pH
`7 · 3, to a concentration of7 x 106 cells/ml. Granulocytes and lymphocytes were incubated for 15 min
`at 37°C in PBS or PBS with 10 J.tg/ml of doxycycline. Irradiation was carried out at 20°C in 10-mm
`diameter test tubes, using a photochemotherapy unit PUV A (H. Haldmann, D-722 Schwenningen,
`FRG) containing 14 fiuorescent tubes (F8T5/BL PUVA, Sylvania) in a bank. About 70% of the
`emission energy ofthese lamps is between 340 and 380 nm. The light intensity is 66 Wjm2 at sample
`Ievel as measured with a UDT model x 80 optometer equipped with a radiometric filter (United
`Detector Technology, lnc., Santa Monica, California, USA). After 30 min irradiation the cells were
`centrifuged, resuspended in PBS to a concentration of 4 x 106 cells/ml, and the EA-RFC testwas
`performed.
`Statistics. The statistical significance of the observed differences was established by Wilcoxon
`tests on unpaired and paired samples as appropriate.
`
`RESULTS
`The pre-treatment Ievels of Fcy-R and C3b-R bearing PMN or lymphocytes from acne patients
`were not significantly different from those of the controls (Table 1). During treatment, however,
`PMN Fcy-R percentages decreased to a mean of57% (P<0·05). Lymphocyte Fcy-R Ievels were not
`significantly affected, nor were the proportians ofT lymphocytes (E-RFC). The percentage of C3 b-
`R bearing cells did not differ from controls.
`Incubation of PMN in increasing concentrations of doxycycline resulted in a significant
`decrease in PMN Fcy-R bearing cell percentage (Fig. 1 ). The Ievels of Fcy-R bearing lymphocytes
`also decreased during incubation, but less markedly.
`
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`
`

`
`A. Nass et al.
`312
`Table 1. Effect of oral doxycycline treatment on leucocyte membrane receptors
`
`Receptor-bearing
`granulocytes (%)
`
`Receptor-bearing
`lymphocytes (%)
`
`No.
`
`Fcy
`
`C3b
`
`E active
`
`E
`
`Fcy
`
`C3b
`
`Acne patients before treatment
`Ance patients during treatment
`Controls
`
`8
`8
`25
`
`80±7
`57±30*
`81 ± 12
`
`69±28
`80±6
`74±17
`
`34±12
`39±13
`39± 12
`
`67±14 32±17
`61± 17 28±18
`66± 11 29±9
`
`38± 12
`43±9
`35± 12
`
`Results are mean values ± s.d.
`*P<0·05
`
`When PMN or lymphocytes were incubated in 50 f1g doxycyclinefml there was a significant
`decrease of Fcy-R bearing PMN which was counteracted when 4 mM Mg2+ was included in the
`medium (Table 2).
`Incubation in 50 flg doxycyclinefml with or without 4 mM Mg2+ had no significant effect on the
`Fcy-R bearing lymphocyte Ievels.
`After PUV A irradiation, granulocytes and lymphocytes preincubated in 10 flg doxycyclinefml
`showed a significant decrease in Fcy-R bearing cell percentage, which was not observed in cells not
`pre-incubated with doxycycline (Table 3).
`
`\
`~~= ~-----------0 • 100
`
`Doxycycfine concentrotion (14g/mll
`Fig. 1. Percentage of Fe receptor positive cells after incubation of granulocytes ( •) and 1ymphocytes ( o) in PBS
`with and without doxycycline. *=No incubation.
`
`Table 2. In vitro effect of doxycycline on leucocyte Fcy receptors
`
`Fcy receptor bearing
`
`Granulocytes (%) Lymphocytes (%)
`
`Incubation
`None
`PBS
`Doxycycline 50 Jtg/ml
`Doxycycline 50 Jtg/ml
`with 4 mmol Mg2+
`
`74±9
`65± 13*
`45± 16*t
`
`65± 13t
`
`44± 18
`39±14
`37± 10
`
`47± 16
`
`Resu1ts are mean of six experiments ± s.d.
`*P<0·01
`tP<0·01
`
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`
`

`
`Effects of doxycycline on leucocyte membrane receptors
`Table 3. Percentages of Fcy-R bearing cells before and after exposure to PUVA light
`
`Afterirradiation (30 min)
`
`Before irradiation Doxycycline+ Doxycycline-
`
`Granulocytes
`Lymphocytes
`
`57±8
`33±9
`
`30±10
`20±15
`
`64±17
`30±6
`
`Doxycycline+ after 15 min preincubation at 37°C in doxycycline
`(10 JLg/ml) in PBS.
`Doxycycline- after 15 min preincubation at 37°C in PBS without
`doxycycline.
`Results are mean±s.d. (five experiments).
`
`DISCUSSION
`
`Wehave shown that doxycycline in vitro or in vivo is associated with a decrease in the proportion of
`Fcy-R bearing PMN. Untreated acne patients had normal Ievels ofC3b-R and Fcy-R bearingcells,
`which is consistent with the findings of Rebora, Dallegri & Patrone (1979) of normal neutrophil
`functions in such patients. Previous studies by several authors have documented a deleterious effect
`oftetracyclines on several PMN functions (Belsheim et al., 1979; Forsgren et al., 1974; Glette et al.,
`1982). The Fcy-R is important in the process of phagocytosis (Kelmpner & Gallin, 1978), and
`interference with this receptor may explain the impaired capacity of doxycycline-incubated PMN to
`phagocytize (Forsgren et al., 1974). One would expect the effect of doxycycline on the Fcy-R ofthe
`PMN tobe paralleled by similar results regarding the lymphocyte Fcy-R. However, these receptors
`arenot necessarily identical, and Colombatti, Heumann & Moretta (1981) found PMN Fcy-R tobe
`resistant to very high concentrations ofpronase, in contrast to lymphocyte Fcy-R. The mechanism
`by which the Fcy-R Ievels are reduced by doxycycline is not clear, but the divalent cation chelating
`effect of the tetracyclines (Gnarpe & Leslie, 1974) may be of irnportance as the addition of 4 mM
`Mg2+ reversed the Fcy-R decrease caused by 50 Jlg/rnl of doxycycline.
`The decreased percentage of Fcy-R bearing cells after doxycycline incubation cornbined with
`PUV A irradiation (Table 3) suggests photo darnage to the cell rnernbrane as one possible
`rnechanism. This cornbined treatrnent reduced the Ievels of Fcy-R positive granulocytes to about
`one-half of the pre-treatrnent Ievels, whereas irradiation alone had no significant effect.
`Tetracyclines arepotent photosensitizers and patients taking tetracyclines often develop skin
`lesions when exposed to sunshine (Harber, Kochevar & Shalita, 1982). Which subcellular structures
`are darnaged during irradiation of granulocytes in the presence of doxycycline is not known, but
`both the photodarnage to the Fcy-R receptor and the photodarnage to granulocyte locornotion and
`phagocytosis (Sandberg et al., 1984) can be explained by deterioration of the plasma rnernbrane.
`Tetracyclines have also been shown to influence lyrnphocyte functions. Banck & Forsgren (1979)
`found low concentrations of doxycycline in vitro to have an inhibiting effect on the mitogenic
`responsesofT and B lyrnphocytes, as weil as on in vitro antibody production, and protein synthesis
`in unstirnulated lymphocytes. Mice treated with tetracycline have been shown to have a reduced
`capacity to rnount delayed-type hypersensitivity to sheep E (Thong & Ferrante, 1980). The present
`study has not, however, demonstrated any significant difference in the proportion ofFcy-R, C3b-R
`or E-R bearing lymphocytes during doxycycline treatment. Neither had our patients low
`proportions ofFcy-R bearing lymphocytes, as was found by Valmin, Halberg & Redström (1982) in
`patients with severe staphylococcal furunculosis. The essentially unchanged percentage of E-RFC
`during doxycycline treatment does not support the clairn of Pudifin et al. (1978) that tetracycline
`treatrnent causes an increase in lymphocytes bearing bothE-R and surface irnrnunoglobulins.
`
`Exh. 1031
`
`

`
`A. Nass et al.
`The technical assistance of Wenke Trovik is highly appreciated. Indicator cells were kindly provided by the
`Broegelmann Research Laboratory for Microbiology, and by the Department of Microbiology and
`Immunology, the Gade Institute. This work was supported by the Norwegian Research Council for Science and
`the Humanities.
`
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