Effects of antibiotics on human polymorphonuclear leukocyte chemotaxis
`in vitro
`
`Y. Ueyama, M. Misaki, Y. Ishihara, T. Matsumura
`Department of Oral and Maxillofacial Surgery II, Okayama University Dental School, Okayama 700,
`Japan
`
`SUM MARY. The effects produced in vitro by minocycline (MINO), aminobenzyl penicillin (ABPC) and cefotiam
`(CTM) on polymorphonuclear leukocytes (PJ.\f:'l) chemotaxis were investigated in this study using the Boyden
`chamber technique and polarization assay. The following results were obtained: CTM brought about a significant
`increase of PMN chemotaxis at the therapeutic dose (10, 100 ~g/ml), MINO reduced PMN chemotaxis signifi-
`cantly at a concentration as low as 1 ~g/ml and ABPC did not influence PMN chemotaxis at concentrations up
`to 100 ~g/ml in both methods. Furthermore, using a checkerboard assay, it was shown that CTM worked as a
`chemokinetic factor.
`
`INTRODUCTIOl'i
`
`A variety of antibiotics are used in the treatment of
`infectious diseases. In the past, antibiotics have been
`evaluated primarily for antibacterial action (minimum
`inhibitory concentration) and pharmacokinetics in
`the host (concentration of blood levels, transfer to
`lesions). More recently, the effects of antibiotics have
`been evaluated from the viewpoint of host defense
`mechanisms against infection. 1 Some of the most
`interesting studies have focused on the effects of
`antibiotics on the functions of polymorphonuclear
`leukocytes (PMN) which play an important role in
`non-specific defense mechanisms against infection. 2•3
`The PMN functions can be divided into migration,
`phagocytosis and bactericidal activity. Of these,
`migration is regarded as one of the major initial
`protective mechanisms, as demonstrated by patients
`with such leukocyte disorders, who are liable to suffer
`from infectious diseases. 4 In this study, the effects of
`different groups of antibiotics on PMN migration in
`vitro were studied by the polarization assay, to
`observe morphological changes on PMN migration,
`and by the Boyden method, to detect alterations in
`function.
`
`gation at 300 x g for 30 min in a density gradient
`usmg Mono-Poly Resolving Medium
`(Flow
`Laboratories, Australia). The residual red cells were
`lysed with 0.83% ammonium chloride. The PMN
`were then washed twice in Hanks balanced salt solu-
`tion (HBSS) and suspended to the desired concen-
`tration
`in HBSS. Cells
`suspensions prepared
`.contained greater than 95% PMN and the viability
`of PMN was over 98% by trypan blue exclusion.
`
`Polarization assay for polymorphonuclear leukocytes
`PMN suspensions (0.45 ml) adjusted at 2.0 x 106
`cells/ml and 0.45 ml of the antibiotics at desired
`concentrations were mixed and incubated in a water
`bath (37"C) for 15 min. After these mixtures were
`exposed to 0.1 ml of 10- 7 M N-formyl-L-methionyl-
`L-leucyi-L-phenylalanine (FMLP, Sigma Chemical,
`St Louis, MO), they were incubated in a water bath
`( 3TC) for I 0 min, the reaction stopped with 2 mJ of
`10% formalin in HBSS (pH 7.4) and the mixture kept
`in ice water for 30 min. The number of polarized
`PMN were counted in a hemocytometer.
`
`MATERIALS A.'IID METHODS
`
`Preparation of antibiotics
`Three kinds of antibiotics were tested by the polariz-
`ation assay and the Boyden method: minocycline
`(Lederle Japan Ltd, MlNO), aminobenzyl penicillin
`(Meiji Seika Kaisya, Ltd, ABPC) and cefotiam
`(Takeda Chemical Industry Ltd, CTM).
`
`Preparation of polymorphonuclear leukocytes
`PMN were obtained from heparinized venous human
`blood of healthy volunteers by means of centrifu-
`
`Assay for migration of polymorphonuclear leukocytes
`Assays were performed by usc of a multiwell chemo-
`taxis assembly (Neuro Probe, Cabin John, Md,
`USA). 5•6 Each of the weBs on the bottom plate was
`filled with 25 J.d of test specimens dissolved in HBSS
`or 25 J.d of HBSS for control. A polycarbonatc filter
`sheet (Neuro Probe) of 3 J.Lm pore size and about
`10 J..lill thickness was then placed on the bottom plate.
`A gasket and a top plate were fixed in place. 50 J.ll of
`PMN suspension (I 05 cells) was then added to each
`well on the top plate. The whole assembly was
`incubated at 37cc for 60 min in humidified air con-
`taining 5% (VoltVol) C02• After incubation, the filter
`
`96
`
`Dr. Reddy's Laboratories, Ltd., et al.
`V.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1033
`Exh. 1033
`
`

`
`Effccts of antibiotics on human polymorphonuclear leukocyte chemotaxis in ritro
`
`97
`
`was rcmoved, fixed and staincd with Diff-Quick
`(International Reagents Co, Kobe, Japan). Migration
`of PMN from the uppcr weil toward thc bottom one
`containing the test specimens or HBSS for control
`was cstimatcd by counting thc number ofPMN which
`had completely passed through the filter; 20 micro-
`scopic fields on the filter surface adjacent to the
`bottom wclls werc randomly selectcd for counting at
`x 1000 magnification (by combination of a x 100 oi1
`immersion objectivc Jens and a x 10 ocular Jens
`equipped with a microgrid (5 by 5 mm; Olympus
`Optical Co, Tokyo, Japan).
`
`RESULTS
`
`Effcct of antibiotics on PMN polarization
`Figure 1 shows the effect of sevcral antibiotics on
`PMN motility examined with the polarization assay.
`Po1arized cclls induced by fMLP decreased in
`number in thc presence of MTNO. This reduction
`occurred in a dosc-dependcnt manner 7.8% ( 1 f.lgjml ),
`
`(%)
`130
`120
`110
`100
`5 90
`.,
`~ 80
`~ 70
`60
`50
`
`CL
`
`I Q
`Co:1centration
`Fig. l- Effect of antibiotics on PMN polarization. Each value
`rcpresents thc mean ±I standard dcviation of six analyses, whcrc
`cvcry analysiswas madc in triplicatc. The differcncc betwecn
`antihiotics and control was significant at a Ievel of 5% (*) by
`Wilcoxon lest.
`
`100 (.,g/rn.e)
`
`12.4% (10 f.lg/ml) or 37.2% (100 f.lgjml) as compared
`to controls.
`ABPC had no effcct on PMN polarization stimu-
`latcd by FMLP in concentrations of 1, 10 f.lg/ml, but
`had a significant Suppression effect at 100 J.lg/ml.
`CTM incrcased po1arized cclls significantly, evcn
`at a concentration of 1 f.lg/ml, and acceleratcd the
`PMN migration significantly in a dosc dependcnt
`fashion; the polarization rose 6.8% (I f.lg/m1 ), 14.3%
`(10 f.lgfm1) or 22.9% (100 f.lg/ml) of control Ievels in
`the clinical dose rangc.
`
`Effect of antibiolies on PMN migration
`Figure 2 shows the result of the PMN migration in
`rcsponse to antibiotics.
`MINO significantly inhibited PMN migration at a
`therapcutically attainab1e concentration (IJ.lg/m1 ).
`At higher concentrations (10, 100 f.lg/m1), significant
`inhibition of PMN migration was effected in a dose-
`dcpcndent manner (Fig. 2A).
`ABPC did not significant1y affcct PM~ migration
`within the clinical dose range of 1 to I 00 J.lg/ml
`(Fig. 28).
`CTM slightly increased PMN migration at a con-
`I f.lg/m1,
`but not
`significantly.
`centration of
`Conccntrations of 10 and 100 f.lg/m1 of CTM, within
`the clinical dosc range, causcd significant enhancc-
`ment of PMN migration (Fig. 2C).
`
`Checkerboard assay with CTM
`Todetermine whether the increase ofPMN migration
`caused by CTM was merely because of increased
`random migration of PMN(chcmokinesis), or
`directed migration in responsc to a conccntration
`gradicnt of CTM(chemotaxis), various concentration
`of CTM were added to eithcr the upper weil, thc
`Jower weil, or both weil in a checkerboard pattern
`(Tab1e I), and migration in these wells were com-
`pared. Whcn there was no differencc in CTM concen-
`tration between thc upper and lower compartments,
`
`(%)
`150
`
`~ 100
`"0
`.!:
`c
`0
`'fii 0,
`
`~
`
`*
`
`*
`
`*
`
`(%)
`150
`
`~ '180
`"0
`.!:
`c
`0 'fii
`0,
`~ 50
`
`-n-----
`
`1
`
`(%)
`150-
`
`*
`
`*
`
`0'-..1.-'-..!.-'-....L....'------
`1 oc
`1 0
`(IJg/mQ)
`10
`100
`c
`A
`B
`Goncentration
`Goncentration
`Goncentration
`Fig. 2- Effcct of antibiotics on P\1N migration. A) MJNO B) ABPC C) CTM. Each value reprcscnts thc mean ±I standard deviation of
`six analyses. where evcry analysiswas made in triplicatc. The dilferencc betwecn antibiotics and control was significant at a Ievel of 5% (*)
`hy Wilcoxon test.
`
`(~."g/rnQ)
`
`10
`
`100 (IJg/mQ)
`
`Exh. 1033
`
`

`
`98
`
`British Journal of Oral and Maxillofacial Surgcry
`
`Table 1- Checkerboard assay of thc ctfect of CTM on P\1~
`migration. The nurober of PMN on the lowcr surface of a
`polycarbonatc liltcr was countcd in triplicate 20 microscopic liclds
`to determinc ± I standard dcviation
`
`Conc. {l.tgiml)
`of cefotiam in
`lower weil
`
`1\ umbcr of cells (mean ± SD) with incrcasing
`conc. (~tg:'ml) of ccfotiam in uppcr weil
`
`0
`
`10
`
`100
`
`0
`I
`10
`100
`
`48.9±7.9
`55.9±3.3
`59.1 ±3.8
`63.3:::2.2
`
`57.7±4.3
`67.3 ±8.0
`67.6±4.8
`71.6±4.7
`
`71.7±5.6
`68.9 ±5.0
`74.6 = 1.3
`82.8 :i: 8.2
`
`82.3±4.4
`79.1±3.7
`82.0±4.9
`95.0±4.8
`
`PMN migration was enhanced in dose-dependent
`fashion. Thc cnhanced migration also oceurred when
`thc concentration of CTM in the upper compartment
`exceeded that in the Iower compartment. The assay
`result strongly suggested that the increased PMN
`migration caused by CTM was due to chemokinesis.
`
`DISCUSSION
`
`The Boyden method measures the numbcr of migrat-
`ing PMN and their migration distances. In contrast,
`the polarization assay records morphological changes
`rclated to function, and therefore, the results may
`represent ehemokinesis of PMN or its initial migrat-
`ing rcaction. 7
`The Boyden method calculates the number of PMN
`which, in accordance with the concentration gradient
`of migration factors, pass through a millipore filter
`and migrate to thc lower surface of filter. Various
`methods have been used to investigate lhe etfects of
`antibiotics on PMN migration. 8- 10 In our study we
`placed an antibiotic in the lower chamber and a PMN
`suspension in the upper in order to eliminate any
`etfects by factors other than the antibiotic and also
`to investigate if the antibiotic itself acts as a chemo-
`tactic factor.
`In I 974, Martin et al 11 reported that, by the Boyden
`I 0 mg/ml inhibited
`method, tetracycline(TC) at 0.1
`PMN migration by 40-50%. Since then, various
`investigators have reported the etfects of various
`antibiotics on PMN function. With respect to protein
`synthesis inhibitors, if selective toxicity of an inhibitor
`is quantitative, it may reduce the migrating function
`of Ieukocytes or inhibit protective function when
`administered in !arge doses. The reports on tetracy-
`cline-based antibiotics and protein synthesis inhibi-
`tors showed similar inhibition of the PMN migrating
`function in vitro. However, since most studies used
`antibiotics al Ievels much higher than those com-
`monly found in serum during their clinical use, the
`rcsults obtaincd could not be applied to the in vivo
`situation. In our study, both thc Boyden method and
`the polarization assay revealed lhat PMN migration
`\\'as inhibited by MINO at concentrations as low as
`I J..lg/ml, which supports the possibility of inhibilion
`in vivo.
`It has been reported that ß-lactam antibiotics exert
`no etfect on migration, 12 although pcnicillin-based
`
`antibiotics belonging to the ß-Iactam group are
`reported to inhibit migration at high dose. 9 In this
`study, ABPC had no apparent etfect on migration at
`I -100 J..lg/ml ( close to blood Ievels common in clini-
`cal use).
`Cephem antibiotics belonging to the ß-lactam
`al
`high
`concentration
`group
`are
`reported
`( 100 I 000 J..lg.iml) to inhibit migration.U The present
`results reveal a dose-dependent enhancement of
`migration by CTM from doses as low as I J..lg/ml to
`I 00 J..Lg/ml, which are serum Ievels common in clinical
`use. Thus, in contrast to thc report by Milatovic
`( 1983 ), 10 these data reveale that some cephem anti-
`biolies may stimulate PMN migration.
`The Boyden method, Iike the other migration
`assays, does not indicate whether chcmotaxis or
`chemokinesis is involved in the enhanced migration.
`Thc checkerboard method, which observes the etfects
`of a concentration gradient on PMN migration, can
`distinguish one from the other. CTM enhanccd the
`PMN migration even in a negative concentration
`gradient, indicating that CTM acted as a chemoki-
`nctic factor.
`Antibiolies can influence the migration of PMN.
`And sincc the collection of PMN in infectious Iesions
`during an acute phase response is due to the migrating
`function of the cells, thosc agents having a negative
`influence on migration may inhibit the body's protec-
`tive mechanisms by impairing PMN functions. To
`administer these agents to paticnls with PMN dys-
`function may further compromisc their ability to cope
`with injection. Wc have presented evidencc that agenls
`which aceeierate the PMN function exist, such as
`CTM, and this activity should be considered as an
`additional beneficial ctfect to the antibacterial activity
`\•.:hen choosing antibiolies for treatment of infcctions.
`
`References
`I. Machida T. Kiyota H. S)~lergism between defcncc mechanism
`and chemothcrapy. Jpn J Bacteriol 1988: 43: 971 980.
`2. Daschncr FD. Leading article: Antibiolies and host dcfcncc
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`1985: 16: 135-141.
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`
`Exh. 1033
`
`

`
`Effects of antibiolies on human po1ymorphonuclear 1eukocytc chemotaxis in vitro
`
`99
`
`cefmetazo1, cefoxitin and imipencm on po1ymorphonudcar
`lcukocytes. Gen Pharmac 1987; 18:613 ·615.
`11. Martin RR, Warr GA, Couch RB, Yeager H, Knight V. Effcct
`oftetracyclinc on 1eukotaxis. J Infcct Dis 1974; 129: 110-116.
`12. Mi1atovie D. Antibiories and phagocytosis. Eur J C1in
`Mierobiol1983; 2:414 425.
`13. Majeski JA. McClcllan MA, A1exander JW. Effcet of
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`Am Surg 1976; Octobcr: 785-788.
`
`The Authors
`Y. Ucvama DDS, PhD
`Assist~nt Professor
`
`M. Misaki DDS
`Instructor
`Y.Jshihara DOS, PhD
`Associate Professor
`T. :VIatsumura DDS, PhD
`Professor
`Department of Oral and Maxillofacial Surgery I1
`Okayama University Dental Schoo1
`2-5-1 Shikata-cho
`Okayama 700
`Japan
`
`Correspondence and requests for offprints to Y. Ucyarna
`
`Paper received 11 March 1993
`Aecepted 30 July 1993
`
`Exh. 1033